Method for identification of t-cell epitopes and use for preparing molecules with reeduced immunogenicity

ABSTRACT

This invention relates to a novel approach for identification of T-cell epitopes, that give rise to an immune reaction in a living host. By means of this novel method biological compounds can be generated which have a no or at least a reduced immunogenicity when exposed to the immune system of a given species and compared with the relevant non-modified entity. Thus the invention relates also to novel biological molecules, especially proteins and antibodies, obtained by the method according to the invention.

FIELD OF INVENTION

[0001] The invention relates to a novel approach of identifying T-cellepitopes that give rise to an immune reaction in a living hostcomprising calculation of potential T-cell epitope values for MHC ClassII molecule binding sites in a peptide by means of computer-aidedmethods. The invention furthermore relates to methods for preparingbiological molecules, above all proteins and antibodies which elicit animmunogenic response when exposed to a host, preferably a human. Bymeans of this method molecules can be prepared which have no or areduced immunogenicity when exposed to the immune system of a givenspecies and compared with the relevant non-modified entity by reductionor removal of potential T-cell epitopes within the sequence of saidoriginally immunogenic molecules. Thus, the invention relates also tonovel biological molecules obtained by the method according to theinvention.

BACKGROUND OF THE INVENTION

[0002] Therapeutic use of a number of peptides, polypeptides andproteins is curtailed because of their immunogenicity in mammals,especially humans. For example, when murine antibodies are administeredto patients who are not immunosuppressed, a majority of such patientsexhibit an immune reaction to the introduced foreign material by makinghuman anti-murine antibodies (HAMA) (e.g. Schroff, R. W. et al (1985)Cancer Res. 45: 879-885; Shawler, D. L. et al (1985) J. Immunol. 135:1530-1535). There are two serious consequences. First, the patient'santi-murine antibody may bind and clear the therapeutic antibody orimmunoconjugate before it has a chance to bind, for example to a tumor,and perform its therapeutic function. Second, the patient may develop anallergic sensitivity to the murine antibody and be at risk ofanaphylactic shock upon any future exposure to murine immunoglobulin.

[0003] Several techniques have been employed to address the HAMA problemand thus enable the use in humans of therapeutic monoclonal antibodies(see, for example, WO-A-8909622, EP-A-0239400, EP-A-0438310,WO-A-9109967). These recombinant DNA approaches have generally reducedthe mouse genetic information in the final antibody construct whilstincreasing the human genetic information in the final construct.Notwithstanding, the resultant “humanized” antibodies have, in severalcases, still elicited an immune response in patients (Issacs J. D.(1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999)Transplantation 68: 1417-1420).

[0004] A common aspect of these methodologies has been the introductioninto the therapeutic antibody, usually of rodent origin, of amino acidresidues, even significant tracts of amino acid residue sequences,identical to those present in human antibody proteins. For antibodies,this process is possible owing to the relatively high degree ofstructural (and functional) conservatism among antibody molecules ofdifferent species. For potentially therapeutic peptides, polypeptidesand proteins, however, where no structural homologue may exist in thehost species (e.g., human) for the therapeutic protein, such processesare not applicable. Furthermore, these methods have assumed that thegeneral introduction of a human amino acid residue sequence will renderthe re-modeled antibody non-immunogenic. It is known, however, thatcertain short peptide sequences (“T-cell epitopes”) can be releasedduring the degradation of peptides, polypeptides or proteins withincells and subsequently be presented by molecules of the majorhistocompatability complex (MHC) in order to trigger the activation ofT-cells. For peptides presented by MHC Class II, such activation ofT-cells can then give rise to an antibody response by direct stimulationof B-cells to produce such antibodies. Accordingly, it would bedesirable to eliminate potential T-cell epitopes from a peptide,polypeptide or a protein. Even proteins of human origin and with thesame amino acid sequences as occur within humans can still induce animmune response in humans. Notable examples include therapeutic use ofgranulocyte-macrophage colony stimulating factor (Wadhwa, M. et al(1999) Clin. Cancer Res. 5: 1353-1361) and interferon alpha 2 (Russo, D.et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl.J. Med. 318: 1409-1413).

[0005] The elimination of T-cell epitopes from proteins has beenpreviously disclosed (see, for example, WO 98/52976, WO 00/34317). Thegeneral methods disclosed in the prior art comprise the following steps:

[0006] (a) Determining the amino acid sequence of the polypeptide orpart thereof

[0007] (b) Identifying one or more potential T-cell epitopes within theamino acid sequence of the protein by any method including determinationof the binding of the peptides to MHC molecules using in vitro or insilico techniques or biological assays.

[0008] (c) Designing new sequence variants with one or more amino acidswithin the identified potential T-cell epitopes modified in such a wayto substantially reduce or eliminate the activity of the T-cell epitopeas determined by the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays. Such sequencevariants are created in such a way to avoid creation of new potentialT-cell epitopes by the sequence variations unless such new potentialT-cell epitopes are, in turn, modified in such a way to substantiallyreduce or eliminate the activity of the T-cell epitope.

[0009] (d) Constructing such sequence variants by recombinant DNAtechniques and testing said variants in order to identify one or morevariants with desirable properties.

[0010] Other techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides and able to bind toT-cell clones from peripheral blood samples from human or experimentalanimal subjects have been used in the art [Kern, F. et al (1998) NatureMedicine 4:975-978; Kwok, W. W. et al (2001) TRENDS in Immunology 22:583-588] and may also be exploited in an epitope identificationstrategy.

[0011] The potential T-cell epitopes are generally defined as any aminoacid residue sequence with the ability to bind to MHC Class IImolecules. Such potential T-cell epitopes can be measured to establishMHC binding. Implicit in the term “T-cell epitope” is an epitope whichwhen bound to MHC molecules can be recognized by the T-cell receptor,and which can, at least in principle, cause the activation of theseT-cells. It is, however, usually understood that certain peptides whichare found to bind to MHC Class II molecules may be retained in a proteinsequence because such peptides are tolerated by the immune within theorganism into which the final protein is administered.

[0012] The invention is conceived to overcome the practical reality thatsoluble proteins introduced into an autologous host with therapeuticintent, can trigger an immune response resulting in development of hostantibodies that bind to the soluble protein. One example amongst othersis interferon alpha 2 to which a proportion of human patients makeantibodies despite the fact that this protein is produced endogenously[Russo, D. et al (1996) Brit. J. Haem. 94: 300-305; Stein, R. et al(1988) New Engl. J. Med. 318: 1409-1413]

[0013] MHC Class II molecules are a group of highly polymorphic proteinswhich play a central role in helper T-cell selection and activation. Thehuman leukocyte antigen group DR (HLA-DR) are the predominant isotype ofthis group of proteins and the major focus of the present invention.However, isotypes HLA-DQ and HLA-DP perform similar functions, hence thepresent invention is equally applicable to these. MHC HLA-DR moleculesare homo-dimers where each “half” is a hetero-dimer consisting of α andβ chains. Each hetero-dimer possesses a ligand binding domain whichbinds to peptides varying between 9 and 20 amino acids in length,although the binding groove can accommodate a maximum of 9-11 aminoacids. The ligand binding domain is comprised of amino acids 1 to 85 ofthe α chain, and amino acids 1 to 94 of the β chain. DQ molecules haverecently been shown to have an homologous structure and the DP familyproteins are also expected to be very similar. In humans approximately70 different allotypes of the DR isotype are known, for DQ there are 30different allotypes and for DP 47 different allotypes are known. Eachindividual bears two to four DR alleles, two DQ and two DP alleles. Thestructure of a number of DR molecules has been solved and suchstructures point to an open-ended peptide binding groove with a numberof hydrophobic pockets which engage hydrophobic residues (pocketresidues) of the peptide [Brown et al Nature (1993) 364: 33; Stern et al(1994) Nature 368: 215]. Polymorphism identifying the differentallotypes of class II molecule contributes to a wide diversity ofdifferent binding surfaces for peptides within the peptide binding groveand at the population level ensures maximal flexibility with regard tothe ability to recognize foreign proteins and mount an immune responseto pathogenic organisms.

[0014] There is a considerable amount of polymorphism within the ligandbinding domain with distinct “families” within different geographicalpopulations and ethnic groups. This polymorphism affects the bindingcharacteristics of the peptide binding domain, thus different “families”of DR molecules will have specificities for peptides with differentsequence properties, although there may be some overlap. Thisspecificity determines recognition of Th-cell epitopes (Class II T-cellresponse) which are ultimately responsible for driving the antibodyresponse to B-cell epitopes present on the same protein from which theTh-cell epitope is derived. Thus, the immune response to a protein in anindividual is heavily influenced by T-cell epitope recognition which isa function of the peptide binding specificity of that individual'sHLA-DR allotype. Therefore, in order to identify T-cell epitopes withina protein or peptide in the context of a global population, it isdesirable to consider the binding properties of as diverse a set ofHLA-DR allotypes as possible, thus covering as high a percentage of theworld population as possible.

[0015] A principal factor in the induction of an immune response is thepresence within the protein of peptides that can stimulate the activityof T-cell via presentation on MHC class II molecules. In order toeliminate or reduce immunogenicity, it is thus desirable to identify andremove T-cell epitopes from the protein.

[0016] The unmodified biological molecules can be produced byrecombinant technologies, which are per se well known in the art, usinga number of different host cell types.

[0017] However, there is a continued need for analogues of saidbiological molecules with enhanced properties. Desired enhancementsinclude alternative schemes and modalities for the expression andpurification of the said therapeutic, but also and especially,improvements in the biological properties of the protein. There is aparticular need for enhancement of the in vivo characteristics whenadministered to the human subject. In this regard, it is highly desiredto provide the selected biological molecule with reduced or absentpotential to induce an immune response in the human subject. Suchproteins would expect to display an increased circulation time withinthe human subject and would be of particular benefit in chronic orrecurring disease settings such as is the case for a number ofindications for said biological molecule.

SUMMARY OF THE INVENTION

[0018] The present invention relates, therefore, to two general aspects:

[0019] (a) a convenient and effective computational method for theidentification and calculation of T-cell epitopes for a globally diversenumber of MHC Class II molecules and, based on this knowledge, fordesigning and constructing new sequence variants of biological moleculeswith improved properties, and

[0020] (b) novel biologically active molecules to be administeredespecially to humans and in particular for therapeutic use; saidbiological molecules are according to this invention immunogeniclymodified polypeptides, proteins or immunoglobulins (antibodies) producedaccording to the method of the invention, whereby the modificationresults in a reduced propensity for the biological molecule to elicit animmune response upon administration to the human subject.

[0021] In particular the invention relates to the modification ofseveral generally well-known proteins and antibodies with hightherapeutic benefit from human or non-human origin obtained by themethod according to the invention to result in proteins that aresubstantially non-immunogenic or less immunogenic than any non-modifiedcounterpart when used in vivo. The molecules modified according to thenovel method of this invention would expect to display an increasedcirculation time within the human subject and would be of particularbenefit in chronic or recurring disease settings such as is the case fora number of indications. The present invention provides for, as specificembodiments and in order to demonstrate the efficacy of the inventivemethod, modified forms of said molecules that are expected to displayenhanced properties in vivo. These molecules with modifiedimmunogenicity, i.e. having a decreased immunogenic potential, can beused in pharmaceutical compositions. Such modified molecules are hereintermed “immunogenicly” modified.

[0022] A method for identifying T-cell epitopes partially by means ofcomputational means can be utilized to calculate theoretical T-cellepitope values and thus identify potential MHC Class II molecule bindingpeptides within a protein; wherein the binding site comprises a sequenceof amino acid sites within the protein. The identified peptides canthereafter be modified without substantially reducing, and possiblyenhancing, the therapeutic value of the protein. This computationalmethod comprises selecting a region of the protein having a known aminoacid residue sequence, sequentially sampling overlapping amino acidresidue segments (windows) of predetermined uniform size and constitutedby at least three amino acid residues from the selected region,calculating MHC Class II molecule binding score for each sampledsegment, and identifying at least one of the sampled segments suitablefor modification, based on the calculated MHC Class II molecule bindingscore for that segment. The overall MHC Class II binding score for thepeptide can then be changed without substantially reducing therapeuticvalue of the protein.

[0023] The MHC Class II molecule binding score for a selected amino acidresidue segment in one aspect of this invention is calculated by summingassigned values for each hydrophobic amino acid residue side chainpresent in the sampled amino acid residue segment of the peptide. Togenerate a graphical overview, the value of that sum can then beassigned to a single amino acid residue at about the midpoint of thesegment. This procedure is repeated for each of the overlapping segments(windows) in the peptide region or regions of interest. The assignedvalue for each aromatic side chain present is about one-half of theassigned value for each hydrophobic aliphatic side chain. Thehydrophobic aliphatic side chains are those present in valine, leucine,isoleucine and methionine. The aromatic side chains are those present inphenylalanine, tyrosine and tryptophan. The preferred assigned value foran aromatic side chain is about 1 and for a hydrophobic aliphatic sidechain is about 2. Other values can be utilized, however.

[0024] Thus, in a first aspect, the invention provides for acomputational-based method suitable for identifying one or morepotential T-cell epitope peptides within the amino acid sequence of abiological molecule by steps including determination of the binding ofsaid peptides to MHC molecules using in vitro or in silico techniques orbiological assays, said method comprises the following steps:

[0025] (a) selecting a region of the peptide having a known amino acidresidue sequence;

[0026] (b) sequentially sampling overlapping amino acid residue segmentsof predetermined uniform size and constituted by at least three aminoacid residues from the selected region;

[0027] (c) calculating MHC Class II molecule binding score for each saidsampled segment by summing assigned values for each hydrophobic aminoacid residue side chain present in said sampled amino acid residuesegment; and

[0028] (d) identifying at least one of said segments suitable formodification, based on the calculated MHC Class II molecule bindingscore for that segment, to change overall MIC Class II binding score forthe peptide without substantially the reducing therapeutic utility ofthe peptide.

[0029] In a specific embodiment, the invention relates to a method,wherein step (c) is carried out by using a Böhm scoring functionmodified to include 12-6 van der Waal's ligand-protein energy repulsiveterm and ligand conformational energy term by

[0030] (1) providing a first data base of MHC Class II molecule models;

[0031] (2) providing a second data base of allowed peptide backbones forsaid MHC Class II molecule models;

[0032] (3) selecting a model from said first data base;

[0033] (4) selecting an allowed peptide backbone from said second database;

[0034] (5) identifying amino acid residue side chains present in eachsampled segment;

[0035] (6) determining the binding affinity value for all side chainspresent in each sampled segment; and optionally

[0036] (7) repeating steps (1) through (5) for each said model and eachsaid backbone.

[0037] In a further embodiment the binding score for each sampledsequence is calculated by (i) providing a first data base of MHC ClassII molecule models; (ii) providing a second data base of allowed peptidebackbones for said MHC Class II molecule models; (iii) providing a thirddatabase of allowed amino acid side chain conformations for each of thetwenty amino acids at each position of each backbone; (iv) selecting amodel from said first data base; (v) selecting an allowed peptidebackbone from said second data base; (vi) identifying amino acid residueside chains present in each sampled segment together with their allowedconformations from said third database; (vii) determining the optimumbinding affinity value for all side chains present in each sampledsegment in each allowed conformation; (viii) repeating steps (v) through(vii) for each said backbone and determining the optimum binding score;and (ix) repeating steps (iv) through (viii) for each said model.

[0038] It should be understood that the three databases described abovecan be combined into one database or any two databases can be combinedto provide a combined database.

[0039] The length of the amino acid residue segments to be sampled canvary. Preferably, the sampled amino acid residue segments areconstituted by about 10 to about 15 amino acid residues, more preferablyabout 13 amino acid residues.

[0040] The sampled amino acid residue segments can be overlapping to avarying degree. Preferably, the sampled amino acid residue segmentsoverlap substantially. Most preferably, consecutive sampled amino acidresidue segments overlap one another by all but one amino acid residue.

[0041] That is, in an amino acid residue segment having n residues, n−1residues are overlapped by the next consecutive sampled amino acidresidue segment.

[0042] Thus, in more detail, the invention relates furthermore to thefollowing further preferred embodiments:

[0043] an accordingly specified method, wherein the assigned value foreach aromatic side chain is about one-half of the assigned value foreach hydrophobic aliphatic side chain;

[0044] an accordingly specified method, wherein the sampled amino acidresidue segment is constituted by 13 amino acid residues;

[0045] an accordingly specified method, wherein consecutive sampledamino acid residue segments overlap by one to five amino acid residues;

[0046] an accordingly specified method, wherein consecutive sampledamino acid residue segments overlap one another substantially;

[0047] an accordingly specified method, wherein all but one of aminoacid residues in consecutive sampled amino acid residue segmentsoverlap.

[0048] In a second basic aspect, the present invention provides,modified forms of different biological molecules with one or more T-cellepitopes removed, wherein said modification may be achieved by themethods described above and in the claims. The molecules can also beproduced by the methods as described in the above-cited prior art,however, the molecules obtained by the methods of this invention showenhanced properties. In the prior art teachings, predicted T-cellepitopes are removed by the use of judicious amino acid substitutionwithin the primary sequence of the therapeutic antibody or non-antibodyprotein of both non-human and human derivation.

[0049] The present invention provides for modified forms of proteins andimmunoglobulins that are expected to display enhanced properties invivo.

[0050] Therefore, it is an object of the invention to provide a methodfor preparing an immunogenicly modified biological molecule derived froma parent molecule, wherein the modified molecule has an amino acidsequence different from that of said parent molecule and exhibits areduced immunogenicity relative to the parent molecule when exposed tothe immune system of a given species; said method comprises: (i)determining the amino acid sequence of the parent biological molecule orpart thereof; (ii) identifying one or more potential T-cell epitopeswithin the amino acid sequence of the protein by any method includingdetermination of the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays, (iii) designing newsequence variants by alteration of at least one amino acid residuewithin the originally identified T-cell epitope sequences, said variantsare modified in such a way to substantially reduce or eliminate theactivity or number of the T-cell epitope sequences and/or the number ofMHC allotypes able to bind peptides derived from said biologicalmolecule as determined by the binding of the peptides to MHC moleculesusing in vitro or in silico techniques or biological assays or bybinding of peptide-MHC complexes to T-cells, (iv) constructing suchsequence variants by recombinant DNA techniques and testing saidvariants in order to identify one or more variants with desirableproperties, and (v) optionally repeating steps (ii)-(iv), wherein theidentification of T-cell epitope sequences according to step (ii) isachieved by a method as specified above and below.

[0051] Specific embodiments of step (iii) according to the inventionrelate to the following summarized steps:

[0052] an accordingly specified method, wherein 1-9 amino acid residuesin any of the originally present T-cell epitope sequences are altered;

[0053] an accordingly specified method, wherein one amino acid residuesin any of the originally present T-cell epitope sequences is altered;

[0054] an accordingly specified method, wherein the amino acidalteration is made with reference to an homologous protein sequence andor to in silico modeling techniques.

[0055] an accordingly specified method, wherein the alteration of theamino acid residues is substitution, deletion or addition of originallypresent amino acid(s) residue(s) by other amino acid residue(s) atspecific position(s).

[0056] an accordingly specified method, wherein additionally furtheralteration, preferably by substitution, addition or deletion of specificamino acid(s), is conducted to restore biological activity of saidbiological molecule.

[0057] With the exception of step (ii) the other steps of the methoddisclosed can be achieved by methods and techniques which are well knownfor skilled workers. Since the modified biological molecules areprepared preferably by recombinant technologies corresponding DNAconstructs which were deduced from the amino acid sequence after havingcompleted the exchange of amino acid residues identified by the methodof step (i). The recombinant techniques used herein are well known inthe art (e.g. Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY, USA).

[0058] The biological molecule obtained according to the invention ispreferably a peptide, a protein, an antibody, an antibody fragment, or afusion protein. The invention includes furthermore modifications,variants, mutations, fragments, derivatives, non-, partially- orcompletely glycosylated forms of said molecules having the same orsimilar biological and/or pharmacological activity.

[0059] Although the method disclosed in this invention is not limited tospecific biological molecules, it is a specific embodiment of theinvention to provide preferably molecules which are known in the art andshow a therapeutic benefit and value. Thus it is a further object of theinvention to provide an immunogenicly modified biological moleculederived from a parent molecule, wherein the modified molecule has anamino acid sequence different from that of said parent molecule andexhibits a reduced immunogenicity relative to the parent molecule whenexposed to the immune system of a given species, obtained by a methodaccording to the invention as disclosed in detail above and below.

[0060] The biological molecules of special interest obtained by saidmethod are selected from the groups:

[0061] (a) monoclonal antibodies:

[0062] anti-40 kD glycoprotein antigen antibody KS ¼,

[0063] anti-GD2 antibody 14.18

[0064] anti-Her2 antibody 4D5 (murine) and humanized version(Herceptin®),

[0065] anti-Her1 (EGFR) antibody c225 and h425

[0066] anti-IL-2R (anti-Tac) antibody (Zenapax®),

[0067] anti-CD52 antibody (CAMPATH®);

[0068] anti-CD20 antibodies (C2B8, Rituxan®; Bexxar®)

[0069] antibody directed to the human C5 complement protein

[0070] (b) human proteins:

[0071] sTNF-R1, sTNF-R2, sTNFR-Fc (Enbrel®),

[0072] protein C, acrp30, ricin A, CNTFR ligands

[0073] subtilisin, GM-CSF, human follicle stimulating hormone (h-fsh)

[0074] β-glucocerebrosidase, GLP-1, apolipoprotein A1,

[0075] leptin (human obesity protein), KGF, G-CSF,

[0076] BDNF, EPO, II-1R antagonist.

[0077] The third basic aspect of the present invention relates to theT-cell epitope sequences that derive from the parent immunogeniclynon-modified biological molecules. These epitopes are preferably 13merpetides. Within these peptides sequences having 9 consecutive amino acidresidues are preferred. Thus it is another object of the invention toprovide access to such epitopes and sequences. In more detail theinvention relates to:

[0078] a use of a potential T-cell epitope peptide within the amino acidsequence of a parent immunogenicly non-modified biological moleculeidentified according to any of the methods as described for preparing abiological molecule with reduced immunogenicity having the samebiological activity;

[0079] a corresponding use of a potential T-cell epitope peptide,wherein said T-cell epitope is a 13mer peptide;

[0080] a use of a peptide sequence consisting of at least 9 consecutiveamino acid residues of a 13mer T-cell epitope as specified above forpreparing a biological molecule with reduced immunogenicity having thesame biological activity as compared with the parent non-modifiedmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081]FIG. 1 is a flow chart illustrating one aspect of the presentcomputational method;

[0082]FIG. 2 is a flow chart illustrating a database generation for acomputational method embodying the present invention;

[0083]FIG. 3 is a flow chart illustrating database interrogation forprofiling a peptide for potential T-cell epitopes;

[0084]FIG. 4 is a further flow chart illustrating the computationalmethod.

[0085]FIG. 5 is a plot of T-cell epitope likelihood index versus aminoacid residue coordinates (positions) of glutamic acid decarboxylase (MW:65000) isoform (GAD 65);

[0086]FIG. 6 is a plot of T-cell epitope likelihood index versus aminoacid residue coordinates (positions) for erythropoietin (EPO);

[0087]FIG. 7 is a plot of T-cell epitope likelihood index versus aminoacid residue coordinates (positions) for humanized anti-A33 monoclonalantibody light chain; and

[0088]FIG. 8 is a plot of T-cell epitope likelihood index versus aminoacid residue coordinates (positions) for humanized anti-A33 monoclonalantibody heavy chain. In the foregoing FIGS. 5-8, the solid line (-)depicts a T-cell epitope index calculated by a computational method inaccordance with the flow chart shown in FIG. 1, and the dotted line(.....) depicts the predicted number of T-cell epitopes calculated inaccordance with the computational method in accordance with the flowchart shown in FIG. 3 according to another aspect of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0089] The term “T-cell epitope” means according to the understanding ofthis invention an amino acid sequence which is able to bind withreasonable efficiency MHC class II molecules (or their equivalent in anon-human species), able to stimulate T-cells and/or also to bind(without necessarily measurably activating) T-cells in complex with MHCclass II. The term “peptide” as used herein and in the appended claims,is a compound that includes two or more amino acids. The amino acids arelinked together by a peptide bond (defined herein below). There are 20different naturally occurring amino acids involved in the biologicalproduction of peptides, and any number of them may be linked in anyorder to form a peptide chain or ring. The naturally occurring aminoacids employed in the biological production of peptides all have theL-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g. up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides and hence the number of differentproteins-that can be formed is practically unlimited.

[0090] The term “less or reduced immunogenic(ity)” used before andthereafter is a relative term and relates to the immunogenicity of therespective original source molecule when exposed in vivo to the sametype of species compared with the molecule modified according to theinvention. The term “modified protein” as used according to thisinvention describes a protein which has reduced number of T-cellepitopes and elicits therefore a reduced immunogenicity relative to theparent protein when exposed to the immune system of a given species. Theterm “non-modified protein” as used according to this inventiondescribes the “parent” protein as compared to the “modified protein” andhas a larger number of T-cell epitopes and, therefore, an enhancedimmunogenicity relative to the modified protein when exposed to theimmune system of a given species.

[0091] “Alpha carbon (Cα)” is the carbon atom of the carbon-hydrogen(CH) component that is in the peptide chain. A “side chain” is a pendantgroup to Cα that can comprise a simple or complex group or moiety,having physical dimensions that can vary significantly compared to thedimensions of the peptide.

[0092] T-cell epitopes can be identified by the computational method ofthe current invention by consideration of amino acid residues importantfor the binding of a particular T-cell epitope to MHC Class IImolecules. Once identified, potential T-cell epitopes can be removed orobliterated from an amino acid residue sequence by alteration, such asmutation, of key amino acid residues in that sequence. Any modificationmade to the sequence of a peptide in a region which is likely to containT-cell epitopes, by deletion, addition or substitution, resulting in arelatively lower overall binding score will have the effect of renderingthe amino acid residue sequence less immunogenic. In some instances, itmay be desirable to enhance the binding of certain peptides to MHC ClassII molecules. For example, it has been proposed that tolerance tocertain autoantigens can be reinstated in individuals suffering fromautoimmune disease if such individuals are treated with peptideanalogues of regions of the autoantigen that are known to contain T-cellepitopes. The natural epitope usually has moderate affinity for MHCClass II molecules, whereas the peptide analogue is made such that ithas a relatively higher affinity for MHC Class II molecules. This highaffinity is important in either promoting immune surveillance to clearsuch T-cells presenting this high affinity epitope, or for them tobecome anergised. This modification to a T-cell epitope can also be madeat the protein level of the peptide, and the entire protein administeredas a therapeutic. There are a number of factors that play importantroles in determining the total structure of a protein or polypeptide.First, the peptide bond, i.e., that bond which joins the amino acids inthe chain together, is a covalent bond. This bond is planar instructure, essentially a substituted amide. An “amide” is any of a groupof organic compounds containing the grouping:

[0093] The planar peptide bond linking Cα of adjacent amino acids may berepresented as depicted below:

[0094] Because the O═C and the C—N atoms lie in a relatively rigidplane, free rotation does not occur about these axes. Hence, a planeschematically depicted by the interrupted line is sometimes referred toas an “amide” or “peptide plane” plane wherein lie the oxygen (O),carbon (C), nitrogen (N), and hydrogen (H) atoms of the peptidebackbone. At opposite corners of this amide plane are located the Cαatoms. Since there is substantially no rotation about the O═C and C—Natoms in the peptide or amide plane, a polypeptide chain thus comprisesa series of planar peptide linkages joining the Cα atoms.

[0095] A second factor that plays an important role in defining thetotal structure or conformation of a polypeptide or protein is the angleof rotation of each amide plane about the common Cα linkage. The terms“angle of rotation” and “torsion angle” are hereinafter regarded asequivalent terms. Assuming that the O, C, N, and H atoms remain in theamide plane (which is usually a valid assumption, although there may besome slight deviations from planarity of these atoms for someconformations), these angles of rotation define the N and Rpolypeptide's backbone conformation, i.e., the structure as it existsbetween adjacent residues. These two angles are known as φ and ψ. A setof the angles φ₁, ψ₁, where the subscript i represents a particularresidue of a polypeptide chain, thus effectively defines the polypeptideThe conventions used in defining the φ, ψ angles, i.e., the referencepoints at which the amide planes form a zero degree angle, and thedefinition of which angle is φ, and which angle is ψ, for a givenpolypeptide, are defined in the literature. See, e.g, Ramachandran etal. Adv. Prot. Chem. 23:283-437 (1968), at pages 285-94, which pages areincorporated herein by reference.

[0096] The present method can be applied to any protein, and is based inpart upon the discovery that in humans the primary Pocket 1 anchorposition of MHC Class II molecule binding grooves has a well designedspecificity for particular amino acid side chains. The specificity ofthis pocket is determined by the identity of the amino acid at position86 of the beta chain of the MHC Class II molecule. This site is locatedat the bottom of Pocket 1 and determines the size of the side chain thatcan be accommodated by this pocket (Marshall, K. W., (1994), J.Immunol., 152:4946-4956). If this residue is a glycine, then allhydrophobic aliphatic and aromatic amino acids (hydrophobic aliphaticsbeing: valine, leucine, isoleucine, methionine and aromatics being:phenylalanine, tyrosine and tryptophan) can be accommodated in thepocket, a preference being for the aromatic side chains. If this pocketresidue is a valine, then the side chain of this amino acid protrudesinto the pocket and restricts the size of peptide side chains that canbe accommodated such that only hydrophobic aliphatic side chains can beaccommodated. Therefore, in an amino acid residue sequence, wherever anamino acid with a hydrophobic aliphatic or aromatic side chain is found,there is the potential for a MHC Class II restricted T-cell epitope tobe present. If the side-chain is hydrophobic aliphatic, however, it isapproximately twice as likely to be associated with a T-cell epitopethan an aromatic side chain (assuming an approximately even distributionof Pocket 1 types throughout the global population).

[0097] A computational method embodying the present invention profilesthe likelihood of peptide regions to contain T-cell epitopes as follows:

[0098] (1) The primary sequence of a peptide segment of predeterminedlength is scanned, and all hydrophobic aliphatic and aromatic sidechains present are identified. (2) The hydrophobic aliphatic side chainsare assigned a value greater than that for the aromatic side chains;preferably about twice the value assigned to the aromatic side chains,e.g., a value of 2 for a hydrophobic aliphatic side chain and a value of1 for an aromatic side chain. (3) The values determined to be presentare summed for each overlapping amino acid residue segment (window) ofpredetermined uniform length within the peptide, and the total value fora particular segment (window) is assigned to a single amino acid residueat an intermediate position of the segment (window), preferably to aresidue at about the midpoint of the sampled segment (window). Thisprocedure is repeated for each sampled overlapping amino acid residuesegment (window). Thus, each amino acid residue of the peptide isassigned a value that relates to the likelihood of a T-cell epitopebeing present in that particular segment (window). (4) The valuescalculated and assigned as described in Step 3, above, can be plottedagainst the amino acid coordinates of the entire amino acid residuesequence being assessed. (5) All portions of the sequence which have ascore of a predetermined value, e.g., a value of 1, are deemed likely tocontain a T-cell epitope and can be modified, if desired. Thisparticular aspect of the present invention provides a general method bywhich the regions of peptides likely to contain T-cell epitopes can bedescribed. Modifications to the peptide in these regions have thepotential to modify the MHC Class II binding characteristics. Accordingto another aspect of the present invention, T-cell epitopes can bepredicted with greater accuracy by the use of a more sophisticatedcomputational method which takes into account the interactions ofpeptides with models of MHC Class II alleles.

[0099] The computational prediction of T-cell epitopes present within apeptide according to this particular aspect contemplates theconstruction of models of at least 42 MHC Class II alleles based uponthe structures of all known MHC Class II molecules and a method for theuse of these models in the computational identification of T-cellepitopes, the construction of libraries of peptide backbones for eachmodel in order to allow for the known variability in relative peptidebackbone alpha carbon (Cα) positions, the construction of libraries ofamino-acid side chain conformations for each backbone dock with eachmodel for each of the 20 amino-acid alternatives at positions criticalfor the interaction between peptide and MHC Class II molecule, and theuse of these libraries of backbones and side-chain conformations inconjunction with a scoring function to select the optimum backbone andside-chain conformation for a particular peptide docked with aparticular MHC Class II molecule and the derivation of a binding scorefrom this interaction.

[0100] Models of MHC Class II molecules can be derived via homologymodeling from a number of similar structures found in the BrookhavenProtein Data Bank (“PDB”). These may be made by the use ofsemi-automatic homology modeling software (Modeller, Sali A. & BlundellT L., 1993. J. Mol Biol 234:779-815) which incorporates a simulatedannealing function, in conjunction with the CHARMm force-field forenergy minimisation (available from Molecular Simulations Inc., SanDiego, Calif.). Alternative modeling methods can be utilized as well.

[0101] The present method differs significantly from other computationalmethods which use libraries of experimentally derived binding data ofeach amino-acid alternative at each position in the binding groove for asmall set of MHC Class II molecules (Marshall, K. W., et al., Biomed.Pept. Proteins Nucleic Acids, 1(3):157-162) (1995) or yet othercomputational methods which use similar experimental binding data inorder to define the binding characteristics of particular types ofbinding pockets within the groove, again using a relatively small subsetof MHC Class II molecules, and then ‘mixing and matching’ pocket typesfrom this pocket library to artificially create further ‘virtual’ MHCClass II molecules (Sturniolo T., et al., Nat. Biotech, 17(6): 555-561(1999). Both prior methods suffer the major disadvantage that, due tothe complexity of the assays and the need to synthesize large numbers ofpeptide variants, only a small number of MHC Class II molecules can beexperimentally scanned. Therefore the first prior method can only makepredictions for a small number of MHC Class II molecules. The secondprior method also makes the assumption that a pocket lined with similaramino-acids in one molecule will have the same binding characteristicswhen in the context of a different Class II allele and suffers furtherdisadvantages in that only those MHC Class II molecules can be‘virtually’ created which contain pockets contained within the pocketlibrary. Using the modeling approach described herein, the structure ofany number and type of MHC Class II molecules can be deduced, thereforealleles can be specifically selected to be representative of the globalpopulation. In addition, the number of MHC Class II molecules scannedcan be increased by making further models further than having togenerate additional data via complex experimentation.

[0102] The use of a backbone library allows for variation in thepositions of the Cα atoms of the various peptides being scanned whendocked with particular MHC Class II molecules. This is again in contrastto the alternative prior computational methods described above whichrely on the use of simplified peptide backbones for scanning amino-acidbinding in particular pockets. These simplified backbones are not likelyto be representative of backbone conformations found in ‘real’ peptidesleading to inaccuracies in prediction of peptide binding. The presentbackbone library is created by superposing the backbones of all peptidesbound to MHC Class II molecules found within the Protein Data Bank andnoting the root mean square (RMS) deviation between the Cα atoms of eachof the eleven amino-acids located within the binding groove. While thislibrary can be derived from a small number of suitable available mouseand human structures (currently 13), in order to allow for thepossibility of even greater variability, the RMS figure for each C″-αposition is increased by 50%. The average Cα position of each amino-acidis then determined and a sphere drawn around this point whose radiusequals the RMS deviation at that position plus 50%. This sphererepresents all allowed Cα positions.

[0103] Working from the Cα with the least RMS deviation (that of theamino-acid in Pocket 1 as mentioned above, equivalent to Position 2 ofthe 11 residues in the binding groove), the sphere isthree-dimensionally gridded, and each vertex within the grid is thenused as a possible location for a Cα of that amino-acid. The subsequentamide plane, corresponding to the peptide bond to the subsequentamino-acid is grafted onto each of these Cαs and the φ and ψ angles arerotated step-wise at set intervals in order to position the subsequentCα. If the subsequent Cα falls within the ‘sphere of allowed positions’for this Cα than the orientation of the dipeptide is accepted, whereasif it falls outside the sphere then the dipeptide is rejected. Thisprocess is then repeated for each of the subsequent Cα positions, suchthat the peptide grows from the Pocket 1 Cα ‘seed’, until all ninesubsequent Cαs have been positioned from all possible permutations ofthe preceding Cαs. The process is then repeated once more for the singleCα preceding pocket 1 to create a library of backbone Cα positionslocated within the binding groove.

[0104] The number of backbones generated is dependent upon severalfactors: The size of the “spheres of allowed positions”; the fineness ofthe gridding of the “primary sphere” at the Pocket 1 position; thefineness of the step-wise rotation of the φ and ψ angles used toposition subsequent Cαs. Using this process, a large library ofbackbones can be created. The larger the backbone library, the morelikely it will be that the optimum fit will be found for a particularpeptide within the binding groove of an MHC Class II molecule. Inasmuchas all backbones will not be suitable for docking with all the models ofMHC Class II molecules due to clashes with amino-acids of the bindingdomains, for each allele a subset of the library is created comprisingbackbones which can be accommodated by that allele. The use of thebackbone library, in conjunction with the models of MHC Class IImolecules creates an exhaustive database consisting of allowed sidechain conformations for each amino-acid in each position of the bindinggroove for each MHC Class II molecule docked with each allowed backbone.This data set is generated using a simple steric overlap function wherea MHC Class II molecule is docked with a backbone and an amino-acid sidechain is grafted onto the backbone at the desired position. Each of therotatable bonds of the side chain is rotated step-wise at set intervalsand the resultant positions of the atoms dependent upon that bond noted.The interaction of the atom with atoms of side-chains of the bindinggroove is noted and positions are either accepted or rejected accordingto the following criteria: The sum total of the overlap of all atoms sofar positioned must not exceed a pre-determined value. Thus thestringency of the conformational search is a function of the intervalused in the step-wise rotation of the bond and the pre-determined limitfor the total overlap. This latter value can be small if it is knownthat a particular pocket is rigid, however the stringency can be relaxedif the positions of pocket side-chains are known to be relativelyflexible. Thus allowances can be made to imitate variations inflexibility within pockets of the binding groove. This conformationalsearch is then repeated for every amino-acid at every position of eachbackbone when docked with each of the MHC Class II molecules to createthe exhaustive database of side-chain conformations.

[0105] A suitable mathematical expression is used to estimate the energyof binding between models of MHC Class II molecules in conjunction withpeptide ligand conformations which are empirically derived by scanningthe large database of backbone/side-chain conformations described above.Thus a protein is scanned for potential T-cell epitopes by subjectingeach possible peptide of length varying between 9 and 20 amino-acids(although the length is kept constant for each scan) to the followingcomputations: an MHC Class II molecule is selected together with apeptide backbone allowed for that molecule and the side-chainscorresponding to the desired peptide sequence are grafted on. Atomidentity and interatomic distance data relating to a particularside-chain at a particular position on the backbone are collected foreach allowed conformation of that amino-acid (obtained from the databasedescribed above). This is repeated for each side-chain along thebackbone and peptide scores derived using a scoring function. The bestscore for that backbone is retained and the process repeated for eachallowed backbone for the selected model. The scores from all allowedbackbones are compared and the highest score is deemed to be the peptidescore for the desired peptide in that MHC Class II model. This processis then repeated for each model with every possible peptide derived fromthe protein being scanned, and the scores for peptides versus models aredisplayed.

[0106] In the context of the present invention, each ligand presentedfor the binding affinity calculation is an amino-acid segment selectedfrom a peptide or protein as discussed above. Thus, the ligand is aselected stretch of amino acids about 9 to 20 amino acids in lengthderived from a peptide, polypeptide or protein of known sequence. Theterms “amino acids” and “residues” are hereinafter regarded asequivalent terms. The ligand, in the form of the consecutive amino acidsof the peptide to be examined grafted onto a backbone from the backbonelibrary, is positioned in the binding cleft of an MHC Class II moleculefrom the MHC Class II molecule model library via the coordinates of theC″-α atoms of the peptide backbone and an allowed conformation for eachside-chain is selected from the database of allowed conformations. Therelevant atom identities and interatomic distances are also retrievedfrom this database and used to calculate the peptide binding score.Ligands with a high binding affinity for the MHC Class II binding pocketare flagged as candidates for site-directed mutagenesis. Amino-acidsubstitutions are made in the flagged ligand (and hence in the proteinof interest) which is then retested using the scoring function in orderto determine changes which reduce the binding affinity below apredetermined threshold value. These changes can then be incorporatedinto the protein of interest to remove T-cell epitopes. Binding betweenthe peptide ligand and the binding groove of MHC Class II moleculesinvolves non-covalent interactions including, but not limited to:hydrogen bonds, electrostatic interactions, hydrophobic (lipophilic)interactions and Van der Waals interactions. These are included in thepeptide scoring function as described in detail below. It should beunderstood that a hydrogen bond is a non-covalent bond which can beformed between polar or charged groups and consists of a hydrogen atomshared by two other atoms. The hydrogen of the hydrogen donor has apositive charge where the hydrogen acceptor has a partial negativecharge. For the purposes of peptide/protein interactions, hydrogen bonddonors may be either nitrogens with hydrogen attached or hydrogensattached to oxygen or nitrogen. Hydrogen bond acceptor atoms may beoxygens not attached to hydrogen, nitrogens with no hydrogens attachedand one or two connections, or sulphurs with only one connection.Certain atoms, such as oxygens attached to hydrogens or imine nitrogens(e.g. C═NH) may be both hydrogen acceptors or donors. Hydrogen bondenergies range from 3 to 7 Kcal/mol and are much stronger than Van derWaal's bonds, but weaker than covalent bonds. Hydrogen bonds are alsohighly directional and are at their strongest when the donor atom,hydrogen atom and acceptor atom are co-linear. Electrostatic bonds areformed between oppositely charged ion pairs and the strength of theinteraction is inversely proportional to the square of the distancebetween the atoms according to Coulomb's law. The optimal distancebetween ion pairs is about 2.8 Å. In protein/peptide interactions,electrostatic bonds may be formed between arginine, histidine or lysineand aspartate or glutamate. The strength of the bond will depend uponthe pKa of the ionizing group and the dielectric constant of the mediumalthough they are approximately similar in strength to hydrogen bonds.

[0107] Lipophilic interactions are favorable hydrophobic-hydrophobiccontacts that occur between he protein and peptide ligand. Usually,these will occur between hydrophobic amino acid side chains of thepeptide buried within the pockets of the binding groove such that theyare not exposed to solvent. Exposure of the hydrophobic residues tosolvent is highly unfavorable since the surrounding solvent moleculesare forced to hydrogen bond with each other forming cage-like clathratestructures. The resultant decrease in entropy is highly unfavorable.Lipophilic atoms may be sulphurs that are neither polar nor hydrogenacceptors and carbon atoms that are not polar.

[0108] Van der Waal's bonds are non-specific forces found between atomswhich are 3-4 Å apart. They are weaker and less specific than hydrogenand electrostatic bonds. The distribution of electronic charge around anatom changes with time and, at any instant, the charge distribution isnot symmetric. This transient asymmetry in electronic charge induces asimilar asymmetry in neighboring atoms. The resultant attractive forcesbetween atoms reaches a maximum at the Van der Waal's contact distancebut diminishes very rapidly at about 1 Å to about 2 Å. Conversely, asatoms become separated by less than the contact distance, increasinglystrong repulsive forces become dominant as the outer electron clouds ofthe atoms overlap. Although the attractive forces are relatively weakcompared to electrostatic and hydrogen bonds (about 0.6 Kcal/mol), therepulsive forces in particular may be very important in determiningwhether a peptide ligand may bind successfully to a protein.

[0109] In one embodiment, the Böhm scoring function (SCORE1 approach) isused to estimate the binding constant. (Böhm, H. J., J. Comput AidedMol. Des., 8(3):243-256 (1994) which is hereby incorporated in itsentirety). In another embodiment, the scoring function (SCORE2 approach)is used to estimate the binding affinities as an indicator of a ligandcontaining a T-cell epitope (Böhm, H. J., J. Comput Aided Mol. Des.,12(4):309-323 (1998) which is hereby incorporated in its entirety).However, the Böhm scoring functions as described in the above referencesare used to estimate the binding affinity of a ligand to a protein whereit is already known that the ligand successfully binds to the proteinand the protein/ligand complex has had its structure solved, the solvedstructure being present in the Protein Data Bank (“PDB”). Therefore, thescoring function has been developed with the benefit of known positivebinding data. To allow for discrimination between positive and negativebinders, a repulsion term can optionally be added to the equation. Inaddition, a more satisfactory estimate of binding energy is achieved bycomputing the lipophilic interactions in a pairwise manner rather thanusing the area based energy term of the above Böhm functions. Therefore,in a preferred embodiment, the binding energy is estimated using amodified Böhm scoring function. In the modified Böhm scoring function,the binding energy between protein and ligand (ΔG_(bind)) is estimatedconsidering the following parameters: The reduction of binding energydue to the overall loss of translational and rotational entropy of theligand (ΔG₀); contributions from ideal hydrogen bonds (ΔG_(hb)) where atleast one partner is neutral; contributions from unperturbed ionicinteractions (ΔG_(ionic)); lipophilic interactions between lipophilicligand atoms and lipophilic acceptor atoms (ΔG_(lipo)); the loss ofbinding energy due to the freezing of internal degrees of freedom in theligand, i.e., the freedom of rotation about each C—C bond is reduced(ΔG_(rot)); the energy of the interaction between the protein and ligand(Δ_(VdW)). Consideration of these terms gives equation 1:

(ΔG _(bind))=(ΔG ₀)+(ΔG_(hb) ×N _(hb))+(ΔG _(ionic) ×N _(ionic))+(ΔG_(lipo)×N_(lipo))+(ΔG _(rot) +N _(rot))+(E_(VdW)).

[0110] Where N is the number of qualifying interactions for a specificterm and, in one embodiment, ΔG₀, ΔG_(hb), ΔG_(ionic), ΔG_(lipo) andΔG_(rot) are constants which are given the values: 5.4, 4.7, 4.7, −0.17,and 1.4, respectively.

[0111] The term N_(hb) is calculated according to equation 2:

Nhb=Σ_(h-bonds) f(ΔR, Δα)×f(N _(neighb))×f _(pcs)

[0112] f(ΔR, Δα) is a penalty function which accounts for largedeviations of hydrogen bonds from ideality and is calculated accordingto equation 3:

f(ΔR,Δ−□)=f1(ΔR)×f2(Δα)

[0113] Where:

[0114] f1(ΔR)=1 if ΔR<=TOL

[0115] or =1−(ΔR−TOL)/0.4 if ΔR<=0.4+TOL

[0116] or ═O if AR>0.4+TOL

[0117] And:

[0118] f2(Δα)=1 if Δα<30°

[0119] or =1−(Δα−30)/50 if Δα<=800

[0120] or =0 if Δα>80°

[0121] TOL is the tolerated deviation in hydrogen bond length=0.25 Å

[0122] ΔR is the deviation of the H—O/N hydrogen bond length from theideal value=1.9 Å

[0123] Δα is the deviation of the hydrogen bond angle∠_(N/O—H . . . O/N) from its idealized value of 180° f(N_(neighb))distinguishes between concave and convex parts of a protein surface andtherefore assigns greater weight to polar interactions found in pocketsrather than those found at the protein surface.

[0124] This function is calculated according to equation 4 below:

f(N _(neighb))=(N _(neighb) /N _(neighb,0))^(α) where α=0.5

[0125] N_(neighb) is the number of non-hydrogen protein atoms that arecloser than 5 Å to any given protein atom.

[0126] N_(neighb,0) is a constant=25

[0127] f_(pcs) is a function which allows for the polar contact surfacearea per hydrogen bond and therefore distinguishes between strong andweak hydrogen bonds and its value is determined according to thefollowing criteria:

[0128] f_(pcs)=β when A_(polar)/N_(HB)<10 Å²

[0129] or f_(pcs)=1 when A_(polar)/N_(HB)>10 Å²

[0130] A_(polar) is the size of the polar protein-ligand contact surface

[0131] N_(HB) is the number of hydrogen bonds

[0132] β is a constant whose value=1.2

[0133] For the implementation of the modified Böhm scoring function, thecontributions from ionic interactions, ΔG_(ionic), are computed in asimilar fashion to those from hydrogen bonds described above since thesame geometry dependency is assumed.

[0134] The term N_(lipo) is calculated according to equation 5 below:

N _(lipo)=Σ_(IL) f(r _(IL))

[0135] f(r_(1L)) is calculated for all lipophilic ligand atoms, 1, andall lipophilic protein atoms, L, according to the following criteria:

[0136] f(r_(1L))=1 when r_(1L)<=R1f(r_(1L))=(r_(1L)−R1)/(R2−R1) whenR2<r_(1L)>R1

[0137] f(r_(1L))=0 when r_(1L)>=R2

[0138] Where: R1=r₁ ^(vdw)+r_(L) ^(vdw)+0.5

[0139] and R2=R1+3.0

[0140] and r₁ ^(vdw) is the Van der Waal's radius of atom 1

[0141] and r_(L) ^(vdw) is the Van der Waal's radius of atom L

[0142] The term N_(rot) is the number of rotable bonds of the amino acidside chain and is taken to be the number of acyclic sp³-sp³ and Sp3-Sp2bonds. Rotations of terminal —CH₃ or —NH₃ are not taken into account.

[0143] The final term, E_(VdW), is calculated according to equation 6below:

E _(VdW)=ε₁ε₂((r₁ ^(vdw) +r ₂ ^(vdw))¹² /r ¹²−(r ₁ ^(vdw) +r ₂ ^(vdw))⁶/r ⁶), where:

[0144] ε₁ and ε2 are constants dependant upon atom identity

[0145] r₁ ^(vdw)+r₂ ^(vdw) are the Van der Waal's atomic radii

[0146] r is the distance between a pair of atoms.

[0147] With regard to equation 6, in one embodiment, the constants ε1and ε₂ are given the atom values: C: 0.245, N: 0.283, O: 0.316, S:0.316, respectively (i.e. for atoms of Carbon, Nitrogen, Oxygen andSulphur, respectively). With regards to equations 5 and 6, the Van derWaal's radii are given the atom values C: 1.85, N: 1.75, O: 1.60, S:2.00 Å.

[0148] It should be understood that all predetermined values andconstants given in the equations above are determined within theconstraints of current understandings of protein ligand interactionswith particular regard to the type of computation being undertakenherein. Therefore, it is possible that, as this scoring function isrefined further as a result of progress in the field of modeling ofmolecular interactions, these values and constants may change hence anysuitable numerical value that gives the desired results in terms ofestimating the binding energy of a protein to a ligand may be used andthus fall within the scope of the present invention.

[0149] As described above, the scoring function is applied to dataextracted from the database of side-chain conformations, atomidentities, and interatomic distances. For the purposes of the presentdescription, the number of MHC Class II molecules included in thisdatabase is 42 models plus four solved structures. It should be apparentfrom the above descriptions that the modular nature of the constructionof the computational method of the present invention means that newmodels can simply be added and scanned with the peptide backbone libraryand side-chain conformational search function to create additional datasets which can be processed by the peptide scoring function as describedabove. This allows for the repertoire of scanned MHC Class II moleculesto easily be increased, or structures and associated data to be replacedif data are available to create more accurate models of the existingalleles.

[0150] It should be understood that, although the above scoring functionis relatively simple compared to some sophisticated methodologies thatare available, the calculations are performed extremely rapidly. Itshould also be understood that the objective is not to calculate thetrue binding energy per se for each peptide docked in the binding grooveof a selected MHC Class II protein. The underlying objective is toobtain comparative binding energy data as an aid to predicting thelocation of T-cell epitopes based on the primary structure (i.e. aminoacid sequence) of a selected protein. A relatively high binding energyor a binding energy above a selected threshold value would suggest thepresence of a T-cell epitope in the ligand. The ligand may then besubjected to at least one round of amino-acid substitution and thebinding energy recalculated. Due to the rapid nature of thecalculations, these manipulations of the peptide sequence can beperformed interactively within the program's user interface oncost-effectively available computer hardware. Major investment incomputer hardware is thus not required.

[0151] It would be apparent to one skilled in the art that otheravailable software could be used for the same purposes. In particular,more sophisticated software which is capable of docking ligands intoprotein binding-sites may be used in conjunction with energyminimization. Examples of docking software are: DOCK (Kuntz et al., J.Mol. Biol., 161:269-288 (1982)), LUDI (Böhm, H.J., J. Comput Aided Mol.Des., 8:623-632 (1994)) and FLEXX (Rarey M., et al., ISMB, 3:300-308(1995)). Examples of molecular modeling and manipulation softwareinclude: AMBER (Tripos) and CHARMm (Molecular Simulations Inc.). The useof these computational methods would severely limit the throughput ofthe method of this invention due to the lengths of processing timerequired to make the necessary calculations. However, it is feasiblethat such methods could be used as a ‘secondary screen’ to obtain moreaccurate calculations of binding energy for peptides which are found tobe ‘positive binders’ via the method of the present invention.

[0152] The limitation of processing time for sophisticated molecularmechanic or molecular dynamic calculations is one which is defined bothby the design of the software which makes these calculations and thecurrent technology limitations of computer hardware. It may beanticipated that, in the future, with the writing of more efficient codeand the continuing increases in speed of computer processors, it maybecome feasible to make such calculations within a more manageabletime-frame. Further information on energy functions applied tomacromolecules and consideration of the various interactions that takeplace within a folded protein structure can be found in: Brooks, B. R.,et al., J. Comput. Chem., 4:187-217 (1983) and further informationconcerning general protein-ligand interactions can be found in:Dauber-Osguthorpe et al., Proteins 4(1):31-47(1988), which areincorporated herein by reference in their entirety. Useful backgroundinformation can also be found, for example, in Fasman, G. D., ed.,Prediction of Protein Structure and the Principles of ProteinConformation, Plenum Press, New York, ISBN: 0-306 4313-9.

[0153] The present prediction method can be calibrated against a dataset comprising a large number of peptides whose affinity for various MHCClass II molecules has previously been experimentally determined.

[0154] According to a preferred embodiment of the method, any one of thespecific prediction methods described herein, or any othercomputer-based method of predicting peptide-MHC Class II interactionsthat yields numerical scores for each peptide/MHC Class II pair, iscalibrated against a data set comprising a large number of peptideswhose affinity for various MHC Class II molecules has previously beenexperimentally determined. By comparison of calculated versusexperimental data, a cut of value can be determined above which it isknown that all experimentally determined T-cell epitopes are correctlypredicted.

[0155] Specifically, the computer-derived numerical score is calculatedfor each peptide/MHC Class II pair in the data set. The score iscalculated such that a higher score represents an increased probabilityof binding. The lowest computer-based score for a peptide/MHC Class IIpair that is found experimentally to bind is taken to be a cutoff. Allcomputer-based scores that are significantly below this cutoff score areconsidered to represent non-binding peptide/MHC Class II pairs, whilecomputer-based scores above the cutoff represent a potential bindingpeptide/MHC Class II pair. In general for a given computer-based scoringalgorithm, there will be some peptide/MHC Class II combinations thatgive scores above the cutoff but that do not actually bind. Thus, thispreferred embodiment of the method may generate false-positives, butwill never or only rarely generate false negatives.

[0156] This cutoff-based embodiment of the method is particularly usefulwhen a goal is to eliminate, by mutation, most or all of the T-cellepitopes from a protein. Specifically, according to a more preferredembodiment of the method of the invention, most or all of the T-cellepitopes are removed from a protein as follows. The protein sequence isscanned by a computer-based algorithm for potential T-cell epitopes.Each potential T-cell epitope is given a score, with increasing scorescorrelated with higher probability of binding to an MHC Class II. Eachpeptide segment with a score greater than a cutoff is mutated such thatthe score of the mutated segment is less than the cutoff. Mutations arepreferentially chosen that do not reduce the activity of the proteinbelow an activity necessary for a given purpose. A multiply mutatedprotein, lacking most or all of its computer-predicted T-cell epitopes,is designed. Such a multiply mutated protein is termed a “Deimmunizedprotein”.

[0157] The DeImmunized protein is synthesized by standard methods. Forexample, an artificial DNA sequence encoding the DeImmunized protein isassembled from synthetic oligonucleotides, ligated into an expressionvector and functionally linked to elements promoting expression of theDeImmunized protein. The DeImmunized protein is then purified bystandard methods. The resulting DeImmunized protein contains mutatedamino acids such that genuine T-cell epitopes are eliminated. Inaddition, the DeImmunized protein will often contain mutated amino acidsin segments that are predicted by an algorithm to be T-cell epitopes,but that are not in fact T-cell epitopes. However, significantdeleterious consequences do not result from the mutations in the falselypredicted epitopes, because the mutations are chosen to have littleeffect on protein activity. Moreover, deleterious consequences do notresult from the possible introduction of new B cell epitopes into aprotein, because the lack of T-cell epitopes prevents a B cell responseto the modified protein.

[0158] Application of the above-described methodology to variouspeptides which may be considered for DeImmunization, for modificationsto enhance MHC Class II binding for therapeutic purposes, is exemplifiedbelow.

[0159] The invention may be applied to any biological molecule having adefined biological and/or pharmacological activity with substantiallythe same primary amino acid sequences as those disclosed herein andwould include therefore molecules derived by genetic engineering meansor other processes. The term “biological molecule” is used herein formolecules which have a biological function and cause a biological,pharmacological or pharmaceutical effect or activity. Preferably,biological molecules according to the inventions are peptides,polypeptides, proteins. Hereunder proteins, immunoglobulins arepreferred. The invention includes also variants and other modificationof a specific polypeptide, protein, fusion protein, immunoglobulin whichhave in principal the same biological activity and a similar (reduced)immunogenicity. Furthermore fragments of antibodies like sFv, Fab, Fab′,F(ab′)2 and Fc and biologically effective fragments of proteins areincluded. Antibodies from human origin or humanized antibodies show perse lower or no immunogenicity in humans and have no or a lower number ofimmunogenic epitopes compared to non-human antibodies. Neverthelessthere is also a need for de-immunization of such molecules since some ofthem have been shown to elicit a significant immune response in humans.Furthermore antigens which elicit a not desired and too strong immuneresponse can be modified according to the method of the invention andresult in antigens which have a reduced immunogenicity which is howeverstrong enough for using the antigen e.g. as vaccine.

[0160] Some molecules, like leptin, such as identified from othermammalian sources have in common many of the peptide sequences of thepresent disclosure and have in common many peptide sequences withsubstantially the same sequence as those of the disclosed listing. Suchprotein sequences equally therefore fall under the scope of the presentinvention.

[0161] The invention relates to analogues of the biological moleculesaccording to the invention in which substitutions of at least one aminoacid residue have been made at positions resulting in a substantialreduction in activity of or elimination of one or more potential T-cellepitopes from the protein.

[0162] One or more amino acid substitutions at particular points withinany of the potential MHC class II ligands identified in the tables ofthe examples may result in a molecule with a reduced immunogenicpotential when administered as a therapeutic to the human host.Preferably, amino acid substitutions are made at appropriate pointswithin the peptide sequence predicted to achieve substantial reductionor elimination of the activity of the T-cell epitope. In practice anappropriate point will preferably equate to an amino acid residuebinding within one of the hydrophobic pockets provided within the MHCclass II binding groove. Amino acid residues in the peptide at positionsequating to binding within other pocket regions within the MHC bindingcleft are also considered and fall under the scope of the present.

[0163] It is understood that single amino acid substitutions within agiven potential T-cell epitope are the most preferred route by which theepitope may be eliminated. Combinations of substitution within a singleepitope may be contemplated and for example can be particularlyappropriate where individually defined epitopes are in overlap with eachother. Moreover, amino acid substitutions either singly within a givenepitope or in combination within a single epitope may be made atpositions not equating to the “pocket residues” with respect to the MHCclass II binding groove, but at any point within the peptide sequence.All such substitutions fall within the scope of the present.

[0164] Amino acid substitutions other than within the peptidesidentified above may be contemplated particularly when made incombination with substitution(s) made within a listed peptide. Forexample a change may be contemplated to restore structure or biologicalactivity of the variant molecule. Such compensatory changes and changesto include deletion or addition of particular amino acid residues fromthe molecule according to the invention resulting in a variant withdesired activity and in combination with changes in any of the disclosedpeptides fall under the scope of the present.

[0165] In another aspect, the present invention relates to nucleic acidsencoding said biological molecules having reduced immunogenicity.Methods for making gene constructs and gene products are well known inthe art. In a final aspect the present invention relates topharmaceutical compositions comprising said biological moleculesobtainable by the methods disclosed in the present invention, andmethods for therapeutic treatment of humans using the modified moleculesand pharmaceutical compositions. As can be seen from the followingexamples, the computational methods described herein above provide avery good indicator of where T-cell epitopes are likely to be found inany peptide. This, therefore, allows identification of regions of aminoacid residue sequences which, if altered by one or more amino acidresidue changes, have the effect of removing T-cell epitopes and thusenhance the therapeutic value of the peptide. By means of this methodbiological molecules like peptides, proteins, immunoglobulins and fusionproteins and the like having enhanced properties and pharmacologicalvalue can be prepared.

[0166] The foregoing description and the examples are intended asillustrative, and are not to be taken as limiting. Still other variantswithin the spirit and scope of this invention are possible and willreadily present themselves to those skilled in the art.

EXAMPLE 1

[0167] This example shows the T-cell epitope likelihood profile of theautoantigen glutamic acid decarboxylase isoform (GAD 65; MW: 65.000),which is involved in the development of Type I diabetes. This particularprotein could be a potential target for increasing the affinity ofT-cell epitopes, and also provides a good example for demonstrating theT-cell epitope likelihood index since it is a relatively long peptide(585 amino acid residues) and, therefore, provides a relatively largesample size for profiling.

[0168] Shown in FIG. 5 is the T-cell epitope likelihood profile for GAD65. The solid line represents the T-cell epitope index calculated usingthe computational method shown in FIG. 1, and the dotted line representsthe T-cell epitope index predicted using the computational method shownin FIGS. 3 and 4.

EXAMPLE 2

[0169] This example shows the T-cell epitope likelihood profile oferythropoietin (EPO), a 193 amino acid residue long cytokine widely usedas an intravenously (IV) administered drug to boost red blood cellcounts. This represents a good example of a biologic drug withtherapeutic value but which could induce inappropriate or undesirableimmune responses, especially with the IV route of administration beingused, and which may, therefore, benefit from de-immunization afterpotential T-cell epitopes therein have been identified.

[0170] Shown in FIG. 6 is the T-cell epitope likelihood profile for EPO.The solid line represents the T-cell epitope index calculated using thecomputational method shown in FIG. 1, and the dotted line indicatesT-cell epitope index predicted using the computational method shown inFIGS. 3 and 4.

EXAMPLE 3

[0171]FIGS. 7 and 8 show the T-cell epitope index for the heavy andlight chains of a mouse humanized monoclonal antibody directed againstA33 antigen. The latter is a transmembrane glycoprotein expressed on thesurface of >95% bowel cancers and, therefore, has potential as ananti-cancer therapeutic.

[0172] In FIGS. 7 and 8, the solid line represents the T-cell epitopeindex calculated using the computational method shown in FIG. 1, and thedotted line represents the T-cell epitope index predicted using thecomputational method shown in FIGS. 3 and 4.

EXAMPLE 4 Leptin

[0173] One of these therapeutically valuable molecules is human obesityprotein, called “leptin”. Leptin is a secreted signaling protein of 146amino acid residues involved in the homeostatic mechanisms maintainingadipose mass (e.g. WO 00/40615, WO 98/28427, WO 96/05309). The protein(and its antagonists) offers significant therapeutic potential for thetreatment of diabetes, high blood pressure and cholesterol metabolism.The protein can be produced by recombinant technologies using a numberof different host T-cell types. The amino acid sequence of leptin(depicted as one-letter code) is as follows:VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

[0174] An amino acid sequence which is part of the sequence of animmunogenically non-modified human obesity protein (leptin) and has apotential MHC class II binding activity is selected from the followinggroup identified according to the method of the invention:VPIQKVQDDTKTL, QKVQDDTKTLIKT, KTLIKTIVTRIND, TLIKTIVTRINDI,KTIVTRINDISHT, TIVTRINDISHTQ, TRINDISHTQSVS, NDISHTQSVSSKQ,QSVSSKQKVTGLD, SSKQKVTGLDFIP, QKVTGLDFIPGLH, TGLDFIPGLHPIL,LDFIPGLHPILTL, DFIPGLHPILTLS, PGLHPILTLSKMD, GLHPILTLSKMDQ,HPILTLSKMDQTL, PILTLSKMDQTLA, LTLSKMDQTLAVY, SKMDQTLAVYQQI,QTLAVYQQILTSM, LAVYQQILTSMPS, AVYQQILTSMPSR, QQILTSMPSRNVI,QILTSMPSRNVIQ, TSMPSRNVIQISN, SRNVIQISNDLEN, RNVIQISNDLENL,NVIQISNDLENLR, IQISNDLENLRDL, NDLENLRDLLHVL, LENLRDLLHVLAF,ENLRDLLHVLAFS, RDLLHVLAFSKSC, DLLHVLAFSKSCH, LHVLAFSKSCHLP,HVLAFSKSCHLPW, LAFSKSCHLPWAS, CHLPWASGLETLD, SGLETLDSLGGVL,DSLGGVLEASGYS, SLGGVLEASGYST, GGVLEASGYSTEV, SGYSTEVVALSRL,

[0175] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity.

[0176] Substitutions carried out according to the methods of theinvention leading to the elimination of potential T-cell epitopes ofhuman leptin (WT=wild type) are: Residue WT # residue Substitutions 3 IA C D E C H K N P Q R S T 6 V A C D E G H K N P Q R S T 13 L A C D E G HK N P Q R S T 14 I A C D E G H K N P Q R S T 17 I A C D E C H K N P Q RS T 18 V A C D E C H K N P Q R S T 21 I A C D E G H K N P Q R S T 24 I AC D E G H K N P Q R S T 30 V A C D E G H K N P Q R S T 36 V A C D E G HK N P Q R S T 39 L A C D E G H K N P Q R S T 41 F A C D E G H K N P Q RS T 42 I A C D E C H K N P Q R S T 45 L A C D E G H K N P Q R S T 48 I AC D E G H K N P Q R S T 49 L A C D E G H K N P Q R S T 51 L A C D E G HK N P Q R S T 54 M A C D E C H K N P Q R S T 58 L A C D E G H K N P Q RS T 60 V A C D E G H K N P Q R S T 61 Y A C D E G H K N P Q R S T 64 I AC D E G H K N P Q R S T 65 L A C D E G H K N P Q R S T 68 M A C D E G HK N P Q R S T 73 V A C D E G H K N P Q R S T 74 I A C D E G H K N P Q RS T 76 I A C D E G H K N P Q R S T 80 L A C D E G H K N P Q R S T 83 L AC D E G H K N P Q R S T 86 L A C D E G H K N P Q R S T 87 L A C D E G HK N P Q R S T 89 V A C D E G H K N P Q R S T 90 L A C D E G H K N P Q RS T 92 F A C D E G H K N P Q R S T 98 L A C D E G H K N P Q R S T 100 WA C D E G H K N P Q R S T 104 L A C D E G H K N P Q R S T 107 L A C D EG H K N P Q R S T 110 L A C D E G H K N P Q R S T 113 V A C D E G H K NP Q R S T 114 L A C D E G H K N P Q R S T 119 Y A C D E G H K N P Q R ST 123 V A C D E G H K N P Q R S T 124 V A C D E G H K N P Q R S T 126 LA C D E G H K N P Q R S T 129 L A C D E G H K N P Q R S T 133 L A C D EG H K N P Q R S T 136 M A C D E G H K N P Q R S T

EXAMPLE 5 Il-1R Antagonist

[0177] IL-1 is an important inflammatory and immune modulating cytokinewith pleiotropic effects n a variety of tissues but may contribute tothe pathology associated with rheumatoid arthritis and other diseasesassociated with local tissue damage. An IL-1 receptor antagonist able toinhibit the action of IL-1 has been purified and the gene cloned[Eisenburg S. P. et al (1990) Nature, 343: 341-346; Carter, D. B. et al(1990) Nature, 344: 633-637]. Others have provided IL-1Ra molecules[e.g. U.S. Pat. No. 5,075,222].

[0178] The amino acid sequence of Il-1Ra (depicted as one-letter code)is as follows:RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

[0179] An amino acid sequence which is part of the sequence of animmunogenically non-modified IL-1Ra which has a potential MHC class IIbinding activity is selected from the following group: RKSSKMQAFRIWD,SKMQAFRIWDVNQ, QAFRIWDVNQKTF, FRIWDVNQKTFYL, RIWDVNQKTFYLR,IWDVNQKTFYLRN, WDVNQKTFYLRNN, KTFYLRNNQLVAG, TFYLRNNQLVAGY,FYLRNNQLVAGYL, LRNNQLVAGYLQG, RNNQLVAGYLQGP, NQLVAGYLQGPNV,QLVAGYLQGPNVN, LVAGYLQGPNVNL, AGYLQGPNVNLEE, GYLQGPNVNLEEK,PNVNLEEKIDVVP, VNLEEKIDVVPIE, EKIDVVPIEPHAL, IDVVPIEPHALFL,DVVPIEPHALFLG, VPIEPHALFLGIH, HALFLGIHGGKMC, ALFLGIHGGKMCL,LFLGIHGGKMCLS, LGIHGGKMCLSCV, GKMCLSCVKSGDE, MCLSCVKSGDETR,SCVKSGDETRLQL, ETRLQLEAVNITD, TRLQLEAVNITDL, LQLEAVNITDLSE,EAVNITDLSENRK, VNITDLSENRKQD, TDLSENRKQDKRF, ENRKQDKRFAFIR,KRFAFIRSDSGPT, FAFIRSDSGPTTS, AFIRSDSGPTTSF, TSFESAACPGWFL,SFESAACPGWFLC, PGWFLCTAMEADQ, WFLCTAMEADQPV, TAMEADQPVSLTN,QPVSLTNMPDEGV, VSLTNMPDEGVMV, TNMPDEGVMVTKF, PDEGVMVTKFYFQ,EGVMVTKFYFQED, GVMVTKFYFQEDE

[0180] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity.

[0181] Substitutions leading to the elimination of potential T-cellepitopes are: Residue WT # Residue Substitution 10 M A C D E Q H K N P QR S T 13 F A C D E G H K N P Q R S T 15 I A C D E G H K N P Q R S T 16 WA C D E G H K N P Q R S T 18 V A C D E G H K N P Q R S T 23 F A C D E GH K N P Q R S T 24 Y A C D E G H K N P Q R S T 25 L A C D E G H K N P QR S T 30 L A C D E G H K N P Q R S T 31 V A C D E G H K N P Q R S T 34 YA C D E G H K N P Q R S T 35 L A C D E Q H K N P Q R S T 40 V A C D E GH K N P Q R S T 42 L A C D E G H K N P Q R S T 46 I A C D E G H K N P QR S T 48 V A C D E G H K N P Q R S T 49 V A C D E G H K N P Q R S T 51 IA C D E G H K N P Q R S T 56 L A C D E G H K N P Q R S T 57 F A C D E GH K N P Q R S T 58 L A C D E G H K N P Q R S T 60 I A C D E G H K N P QR S T 65 M A C D E G H K N P Q R S T 67 L A C D E G H K N P Q R S T 70 VA C D E G H K N P Q R S T 78 L A C D E G H K N P Q R S T 80 L A C D E GH K N P Q R S T 83 V A C D E G H K N P Q R S T 85 I A C D E G H K N P QR S T 88 L A C D E G H K N P Q R S T 98 F A C D E G H K N P Q R S T 100F A C D E G H K N P Q R S T 101 I A C D E G H K N P Q R S T 119 W A C DE G H K N P Q R S T 120 F A C D E G H K N P Q R S T 121 L A C D E G H KN P Q R S T 125 M A C D E G H K N P Q R S T 131 V A C D E G H K N P Q RS T 133 L A C D E G H K N P Q R S T 136 M A C D E G H K N P Q R S T 141V A C D E G H K N P Q R S T 142 M A C D E G H K N P Q R S T

EXAMPLE 6 BDNF

[0182] Another therapeutically valuable molecule is “human brain-derivedneutrophic factor (BDNF)”. BNDF is glycoprotein of the nerve growthfactor family of proteins. The mature 119 amino acid glycoprotein isprocessed from a larger pre-cursor to yield a neutrophic factor thatpromotes the survival of neuronal cell populations [Jones K. R. &Reichardt, L. F. (1990) Proc. Natl. Acad. Sci U.S.A. 87: 8060-8064].Such neuronal cells are all located either in the central nervous systemor directly connected to it. Recombinant preparations of BNDF haveenabled the therapeutic potential of the protein to be explored for thepromotion of nerve regeneration and degenerative disease therapy.

[0183] The amino acid sequence of human brain-derived neutrophic factor(BDNF) (depicted as one-letter code) is as follows:HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWFIRIDTSCVCTLTIKRGR

[0184] Others have provided modified BNDF molecules [U.S. Pat. No.5,770,577] and approaches towards the commercial production ofrecombinant BNDF molecules [U.S. Pat. No. 5,986,070].

[0185] An amino acid sequence which is part of the sequence of animmunogenically non-modified human brain-derived neurotrophic factor(BDNF) and has a potential MHC class II binding activity is selectedfrom the following group: GELSVCDSISEWV, LSVCDSISEWVTA, DSISEWVTAADKK,SEWVTAADKKTAV, EWVTAADKKTAVD, WVTAADKKTAVDM, KTAVDMSGGTVTV,TAVDMSGGTVTVL, VDMSGGTVTVLEK, GTVTVLEKVPVSK, VTVLEKVPVSKGQ,TVLEKVPVSKGQL, EKVPVSKGQLKQY, VPVSKGQLKQYFY, GQLKQYFYETKCN,KQYFYETKCNPMG, QYFYETKCNPMGY, YFYETKCNPMGYT, NPMGYTKEGCRGI,MGYTKEGCRGIDK, RGIDKRHWNSQCR, RHWNSQCRTTQSY, HWNSQCRTTQSYV,QSYVRALTMDSKK, SYVRALTMDSKKR, RALTMDSKKRIGW, LTMDSKKRIGWRF,KRIGWRFIRIDTS, IGWRFIRIDTSCV, GWRFIRIDTSCVC, WRFIFIDTSCVCT,RFIRIDTSCVCTL, IRIDTSCVCTLTI, IDTSCVCTLTIKR

[0186] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity.

[0187] Substitutions leading to the elimination of potential T-cellepitopes of human brain-derived neurophic factor (BDNF) (WT=wild type)are: Residue WT # Residue Substitution 10 L A C D E G H K N P Q R S T 16I A C D E G H K N P Q R S T 20 V A C D E G H K N P Q R S T 29 V A C D EG H K N P Q R S T 31 M A C D E G H K N P Q R S T 36 V A C D E G H K N PQ R S T 38 V A C D E G H K N P Q R S T 39 L A C D E G H K N P Q R S T 42V A C D E G H K N P Q R S T 44 V A C D E G H K N P Q R S T 49 L A C D EG H K N P Q R S T 52 Y A C D E G H K N P Q R S T 53 F A C D E G H K N PQ R S T 54 Y A C D E G H K N P Q R S T 61 M A C D E G H K N P Q R S T 63Y A C D E G H K N P Q R S T 71 I A C D E G H K N P Q R S T 76 W A C D EG H K N P Q R S T 86 Y A C D E G H K N P Q R S T 87 V A C D E G H K N PQ R S T 90 L A C D E G H K N P Q R S T 92 M A C D E G H K N P Q R S T 98I A C D E G H K N P Q R S T 100 W A C D E G H K N P Q R S T 102 F A C DE G H K N P Q R S T 103 I A C D E G H K N P Q R S T 105 I A C D E G H KN P Q R S T

EXAMPLE 7 EPO

[0188] Another therapeutically valuable molecule is erythropoietin(EPO). EPO is a glycoprotein hormone involved in the maturation oferythroid progenitor cells into erythrocytes. Naturally occurring EPO isproduced by the liver during foetal life and by the kidney of adults andcirculates in the blood to stimulate production of red blood cells inbone marrow. Anaemia is almost invariably a consequence of renal failuredue to decreased production of EPO from the kidney. Recombinant EPO isused as an effective treatment of anaemia resulting from chronic renalfailure. Recombinant EPO (expressed in mammalian cells) having the aminoacid sequence 1-165 of human erythropoietin [Jacobs, K. et al (1985)Nature, 313: 806-810; Lin, F.-K. et al (1985) Proc. Natl. Acad. Sci.U.S.A. 82:7580-7585] contains three N-linked and linked oligosaccharidechains each containing terminal sialic acid residues. The latter aresignificant in enabling EPO to evade rapid clearance from thecirculation by the hepatic asialoglycoprotein binding protein.

[0189] The amino acid sequence of EPO (depicted as one-letter code) isas follows:APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

[0190] An amino acid sequence which is part of the sequence of animmunogenically non-modified human erythropoietin (EPO) and has apotential MHC class II binding activity is selected from the followinggroup: PRLICDSRVLERY, RLICDSRVLERYL, ICDSRVLERYLLE, CDSRVLERYLLEA,SRVLERYLLEAKE, RVLERYLLEAKEA, LERYLLEAKEAEN, ERYLLEAKEAENI,RYLLEAKEAENIT, YLLEAKEAENITT, LEAKEAENITTGC, KEAENITTGCAEH,ENITTGCAEHCSL, CSLNENITVPDTK, NENITVPDTKVNF, ENITVPDTKVNFY,NITVPDTKVNFYA, ITVPDTKVNFYAW, TKVNFYAWKRMEV, VNFYAWKRMEVGQ,NFYAWKRMEVGQQ, YAWKRMEVGQQAV, KRMEVGQQAVEVW, RMEVGQQAVEVWQ,MEVGQQAVEVWQG, QAVEVWQGLALLS, AVEVWQGLALLSE, VEVWQGLALLSEA,EVWQGLALLSEAV, VWQGLALLSEAVL, WQGLALLSEAVLR, QGLALLSEAVLRG,LALLSEAVLRGQA, ALLSEAVLRGQAL, LSEAVLRGQALLV, SEAVLRGQALLVN,EAVLRGQALLVNS, AVLRGQALLVNSS, QALLVNSSQPWEP, ALLVNSSQPWEPL,LLVNSSQPWEPLQ, QPWEPLQLHVDKA, EPLQLHVDKAVSG, LQLHVDKAVSGLR,LHVDKAVSGLRSL, KAVSGLRSLTTLL, SGLRSLTTLLRAL, RSLTTLLRALGAQ,SLTTLLRALGAQK, TTLLRALGAQKEA, TLLRALGAQKEAI, RALGAQKEAISPP,AQKEAISPPDAAS, EAISPPDAASAAP, SPPDAASAAPLRT, ASAAPLRTITADT,APLRTITADTFRK, RTITADTFRKLFR, TITADTFRKLFRV, DTFRKLFRVYSNF,RKLFRVYSNFLRG, KLFRVYSNFLRGK, FRVYSNFLRGKLK, RVYSNFLRGKLKL,YSNFLRGKLKLTY, SNFLRGKLKLYTG, NFLRGKLKLYTGE, RGKLKLYTGEACR,GKLKLYTGEACRT, LKLYTGEACRTGD, KLYTGEACRTGDR

[0191] Substitutions leading to the elimination of potential T-cellepitopes of human erythropoietin WT=wild type) are: Residue WT # residueSubstitutions 5 L A C D E G H K N P Q R S T 6 I A C D E G H K N P Q R ST 11 V A C D E G H K N P Q R S T 12 L A C D E G H K N P Q R S T 15 Y A CD E G H K N P Q R S T 16 L A C D E G H K N P Q R S T 17 L A C D E G H KN P Q R S T 25 I A C D E G H K N P Q R S T 35 L A C D E G H K N P Q R ST 39 I A C D E G H K N P Q R S T 41 V A C D E G H K N P Q R S T 46 V A CD E G H K N P Q R S T 48 F A C D E G H K N P Q R S T 49 Y A C D E G H KN P Q R S T 51 W A C D E G H K N P Q R S T 54 M A C D E G H K N P Q R ST 56 V A C D E G H K N P Q R S T 61 V A C D E G H K N P Q R S T 63 V A CD E G H K N P Q R S T 64 W A C D E G H K N P Q R S T 67 L A C D E G H KN P Q R S T 69 L A C D E G H K N P Q R S T 70 L A C D E G H K N P Q R ST 74 V A C D E G H K N P Q R S T 75 L A C D E G H K N P Q R S T 80 L A CD E G H K N P Q R S T 81 L A C D E G H K N P Q R S T 82 N A C D E G H KN P Q R S T 88 W A C D E G H K N P Q R S T 91 L A C D E G H K N P Q R ST 93 L A C D E G H K N P Q R S T 95 V A C D E G H K N P Q R S T 99 V A CD E G H K N P Q R S T 102 L A C D E G H K N P Q R S T 105 L A C D E G HK N P Q R S T 108 L A C D E G H K N P Q R S T 109 L A C D E G H K N P QR S T 112 L A C D E G H K N P Q R S T 119 I A C D E G H K N P Q R S T130 L A C D E G H K N P Q R S T 133 I A C D E G H K N P Q R S T 138 F AC D E G H K N P Q R S T 141 L A C D E G H K N P Q R S T 142 F A C D E GH K N P Q R S T 144 V A C D E G H K N P Q R S T 145 Y A C D E G H K N PQ R S T 148 F A C D E G H K N P Q R S T 149 L A C D E G H K N P Q R S T153 L A C D E G H K N P Q R S T 155 L A C D E G H K N P Q R S T 156 Y AC D E G H K N P Q R S T

EXAMPLE 8 G-CSF

[0192] Granulocyte colony stimulating factor (G-CSF) is an importanthaemopoietic cytokine currently used in treatment of indications wherean increase in blood neutrophils will provide benefits. These includecancer therapy, various infectious diseases and related conditions suchas sepsis. G-CSF is also used alone, or in combination with othercompounds and cytokines in the ex vivo expansion of haemopoeitic cellsfor bone marrow transplantation.

[0193] Two forms of human G-CSF are commonly recognized for thiscytoldne. One is a protein of 177 amino acids, the other a protein of174 amino acids [Nagata et al. (1986), EMBO J. 5: 575-581], the 174amino acid form has been found to have the greatest specific in vivobiological activity. Recombinant DNA techniques have enabled theproduction of commercial scale quantities of G-CSF exploiting botheukaryotic and prokaryotic host cell expression systems.

[0194] The amino acid sequence of human granulocyte colony stimulatingfactor (G-CSF) (depicted as one-letter code) is as follows:TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEK LCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPT LDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRH LAQP.

[0195] Other polypeptide analogues and peptide fragments of G-CSF havebeen previously disclosed, including forms modified by site-specificamino acid substitutions and or by modification by chemical adducts.Thus U.S. Pat. No. 4,810,643 discloses analogues with the particular Cysresidues replaced with another amino acid, and G-CSF with an Ala residuein the first (N-terminal) position. EP 0335423 discloses themodification of at least one amino group in a polypeptide having G-CSFactivity. EP 0272703 discloses G-CSF derivatives having amino acidsubstituted or deleted near the N terminus. EP 0459630 discloses G-CSFderivatives in which Cys 17 and Asp 27 are replaced by Ser residues. EP0 243 153 discloses G-CSF modified by inactivating at least one yeastKEX2 protease processing site for increased yield in recombinantproduction and U.S. Pat. No. 4,904,584 discloses lysine alteredproteins. WO 90/12874 discloses further Cys altered variants andAustralian patent document AU 10948/92 discloses the addition of aminoacids to either terminus of a G-CSF molecule for the purpose of aidingin the folding of the molecule after prokaryotic expression.AU-76380/91, discloses G-CSF variants at positions 50-56 of the G-CSF174 amino acid form, and positions 53-59 of the 177 amino acid form.Additional changes at particular His residues were also disclosed. Anamino acid sequence which is part of the sequence of an immunogenicallynon-modified human granulocyte colony stimulating factor (G-CSF) and hasa potential MHC class II binding activity is selected from the followinggroup: TPLGPASSLPQSF, SSLPQSFLLKCLE, QSFLLKCLEQVRK, SFLLKCLEQVRKI,FLLKCLEQVRKIQ, KCLEQVRKIQGDG, EQVRKIQGDGAAL, RKIQGDGAALQEK,AALQEKLVSECAT, EKLVSECATYKLC, KLVSECATYKLCH, AALQEKLCATYKL,EKLCATYKLCHPE, ATYKLCHPEELVL, YKLCHPEELVLLG, EELVLLGHSLGIP,ELVLLGHSLGIPW, HSLGIPWAPLSSC, IPWAPLSSCPSQA, APLSSCPSQALQL,QALQLAGCLSQLH, GCLSQLHSGLFLY, SQLHSGLFLYQGL, SGLFLYQGLLQAL,GLFLYQGLLQALE, LFLYQGLLQALEG, FLYQGLLQALEGI, QGLLQALEGISPE,GLLQALEGISPEL, QALEGISPELGPT, EGISPELGPTLDT, PTLDTLQLDVADF,DTLQLDVADFATT, LQLDVADFATTIW, LDVADFATTIWQQ, TTIWQQMEELGMA,TIWQQMEELGMAP, QQMEELGMAPALQ, EELGMAPALQPTQ, LGMAPALQPTQGA,PALQPTQGAMPAF, GAMPAFASAFQRR, PAFASAFQRRAGG, SAFQRRAGGVLVA,GGVLVASHLQSFL, GVLVASHLQSFLE, VLVASHLQSFLEV, SHLQSFLEVSYRV,QSFLEVSYRVLRH, SFLEVSYRVLRHL, LEVSYRVLRHLAQ

[0196] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity.

[0197] Substitutions leading to the elimination of potential T-cellepitopes of human granulocyte colony stimulating factor (G-CSF) (WT=wildtype) are: Residue WT # Residue Substitution 3 L A C D E G H K N P O R ST 9 L A C D E G H K N P Q R S T 14 L A C D E G H K N P Q R S T 15 L A CD E G H K N P Q R S T 18 L A C D E G H K N P Q R S T 21 V A C D E G H KN P Q R S T 24 I A C D E G H K N P Q R S T 31 L A C D E G H K N P Q R ST 35 L A C D E G H K N P Q R S T 39 Y A C D E G H K N P Q R S T 41 L A CD E G H K N P Q R S T 47 L A C D E G H K N P Q R S T 48 V A C D E G H KN P Q R S T 49 L A C D E G H K N P Q R S T 50 L A C D E G H K N P Q R ST 54 L A C D E G H K N P Q R S T 56 I A C D E G H K N P Q R S T 58 W A CD E G H K N P Q R S T 61 L A C D E G H K N P Q R S T 69 L A C D E G H KN P Q R S T 71 L A C D E G H K N P Q R S T 75 L A C D E G H K N P Q R ST 78 L A C D E G H K N P Q R S T 82 L A C D E G H K N P Q R S T 83 F A CD E G H K N P Q R S T 84 L A C D E G H K N P Q R S T 85 Y A C D E G H KN P Q R S T 88 L A C D E G H K N P Q R S T 89 L A C D E G H K N P Q R ST 92 L A C D E G H K N P Q R S T 95 I A C D E G H K N P Q R S T 99 L A CD E G H K N P Q R S T 103 L A C D E G H K N P Q R S T 106 L A C D E G HK N P Q R S T 108 L A C D E G H K N P Q R S T 110 V A C D E G H K N P QR S T 113 F A C D E G H K N P Q R S T 117 I A C D E G H K N P Q R S T118 W A C D E G H K N P Q R S T 121 M A C D E G H K N P Q R S T 124 L AC D E G H K N P Q R S T 130 L A C D E G H K N P Q R S T 137 M A C D E GH K N P Q R S T 140 F A C D E G H K N P Q R S T 144 F A C D E G H K N PQ R S T 151 V A C D E G H K N P Q R S T 152 L A C D E G H K N P Q R S T153 V A C D E G H K N P Q R S T 157 L A C D E G H K N P Q R S T 160 F AC D E G H K N P Q R S T 161 L A C D E G H K N P Q R S T 163 V A C D E GH K N P Q R S T

EXAMPLE 9 KGF

[0198] Another valuable molecule is keratinocyte growth factor (KGF).KGF is a member of the fibroblast growth factor (FGF)/heparin-bindinggrowth factor family of proteins. It is a secreted glycoproteinexpressed predominantly in the lung, promoting wound healing bystimulating the growth of keratinocytes and other epithelial cells[Finch et al (1989), Science 24: 752-755; Rubin et al (1989), Proc.Natl. Acad. Sci. U.S.A. 86: 802-806]. The mature (processed) form of theglycoprotein comprises 163 amino acid residues and may be isolated fromconditioned media following culture of particular cell lines [Rubin etal, (1989) ibid.], or produced using recombinant techniques [Ron et al(1993) J. Biol. Chem. 268: 2984-2988]. The protein is of therapeuticvalue for the stimulation of epithelial cell growth in a number ofsignificant disease and injury repair settings. This disclosurespecifically pertains the human KGF protein being the mature (processed)form of 163 amino acid residues. Others have also provided KGF molecules[e.g. U.S. Pat. No. 6,008,328; WO90/08771;] including modified KGF [Ronet al (1993) ibid; WO9501434]. However, such teachings have notrecognized the importance of T-cell epitopes to the immunogenicproperties of the protein nor have been conceived to directly influencesaid properties in a specific and controlled way according to the schemeof the present invention.

[0199] The amino acid sequence of keratinocyte growth factor (KGF)(depicted as one-letter code) is as follows:MCNDMTPEQMATNVNCSSPERHTRSYDYMEGGDI RVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLYAKKE CNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHFLPMAIT

[0200] An amino acid sequence which is part of the sequence of animmunogenically non-modified human keratinocyte growth factor (KGF) andhas a potential MHC class II binding activity is selected from thefollowing group: NDMTPEQMATNVN, DMTPEQMATNVNC, EQMATNVNCSSPE,TNVNCSSPERHTR, RSYDYMEGGDIRV, YDYMEGGDIRVRR, DYMEGGDIRVRRL,GDIRVRRLFCRTQ, IRVRRLFCRTQWY, RRLFCRTQWYLRI, RLFCRTQWYLRID,TQWYLRIDKRGKV, QWYLRIDKRGKVK, WYLRIDKRGKVKG, LRIDKRGKVKGTQ,GKVKGTQEMKNNY, QEMKNNYNIMEIR, NNYNIMEIRTVAV, YNIMEIRTVAVGI,NIMEIRTVAVGIV, MEIRTVAVGIVAI, RTVAVGIVAIKGV, VAVGIVAIKGVES,VGIVAIKGVESEF, VAIKGVESEFYLA, KGVESEFYLAMNK, SEFYLAMNKEGKL,EFYLAMNKEGKLY, FYLAMNKEGKLYA, LAMNKEGKLYAKK, GKLYAKKECNEDC,KLYAKKECNEDCN, CNFKELILENHYN, KELILENHYNTYA, ELILENHYNTYAS,LILENHYNTYASA, NHYNTYASAKWTH, NTYASAKWTHNGG, AKWTHNGGEMFVA,GEMFVALNQKGIP, EMFVALNQKGIPV, FVALNQKGIPVRG, VALNQKGIPVRGK,KGIPVRGKKTKKE, IPVRGKKTKKEQK, KTKKEQKTAHFLP

[0201] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity. Substitutions leading to theelimination of potential T-cell epitopes of human keratinocyte growthfactor (KGF) (WT=wild type) are: Residue WT # residue Substitution 5 M AC D E G H K N P Q R S T 10 M A C D E G H K N P Q R S T 14 V A C D E G HK N P Q R S T 26 Y A C D E G H K N P Q R S T 28 Y A C D E G H K N P Q RS T 29 M A C D E G H K N P Q R S T 34 I A C D E G H K N P Q R S T 36 V AC D E G H K N P Q R S T 39 L A C D E G H K N P Q R S T 40 F A C D E G HK N P Q R S T 45 W A C D E G H K N P Q R S T 46 Y A C D E G H K N P Q RS T 47 L A C D E G H K N P Q R S T 49 I A C D E G H K N P Q R S T 55 V AC D E G H K N P Q R S T 61 M A C D E G H K N P Q R S T 65 Y A C D E G HK N P Q R S T 67 I A C D E G H K N P Q R S T 68 M A C D E G H K N P Q RS T 70 I A C D E G H K N P Q R S T 73 V A C D E G H K N P Q R S T 75 V AC D E G H K N P Q R S T 77 I A C D E G H K N P Q R S T 78 V A C D E G HK N P Q R S T 80 I A C D E G H K N P Q R S T 83 V A C D E G H K N P Q RS T 87 F A C D E G H K N P Q R S T 88 Y A C D E G H K N P Q R S T 89 L AC D E G H K N P Q R S T 91 M A C D E G H K N P Q R S T 97 L A C D E G HK N P Q R S T 98 Y A C D E G H K N P Q R S T 109 F A C D E G H K N P Q RS T 112 L A C D E G H K N P Q R S T 113 I A C D E G H K N P Q R S T 114L A C D E G H K N P Q R S T 118 Y A C D E G H K N P Q R S T 121 Y A C DE G H K N P Q R S T 126 W A C D E G H K N P Q R S T 133 M A C D E G H KN P Q R S T 134 F A C D E G H K N P Q R S T 135 V A C D E G H K N P Q RS T 137 L A C D E G H K N P Q R S T 142 I A C D E G H K N P Q R S T 144V A C D E G H K N P Q R S T

EXAMPLE 10 Soluble TNF RI

[0202] The sTNF-RI (soluble tumor necrosis factor receptor type I) is aderivative of the human tumor necrosis factor receptor describedpreviously [Gray, P. W. et al (1990) Proc. Nat. Acad. Sci. U.S.A. 87:7380-7384; Loetschere, H. et al, (1990) Cell 61: 351-359; Schall, T. J.et al (1990) Cell 61: 361-370], comprising the extracellular domain ofthe intact receptor and exhibiting an approximate molecular weight of 30KDa. Additional soluble TNF inhibitors and in particular a 40 KDa formare also known [U.S. Pat. No. 6,143,866]. The soluble forms are able tobind tumor necrosis factor alpha with high affinity and inhibit thecytotoxic activity of the cytokine in vitro. Recombinant preparations ofsTNF-RI are of significant therapeutic value for the treatment ofdiseases where an excess level of tumor necrosis factor is causing apathogenic effect. Indications such as cachexia, sepsis and autoimmunedisorders including, and in particular, rheumatoid arthritis and othersmay be targeted by such therapeutic preparations of sTNF-RI. Othersincluding Brewer et al., U.S. Pat. No. 6,143,866, have provided modifiedsTNF-RI molecules

[0203] Peptide sequences in a human 30 KDa sTNF-RI with potential humanMHC class II binding activity: DSVCPQGKYIHPQ, KYIHPQNNSICCT,NSICCTKCHKGTY, TYLYNDCPGPGQD, YLYNDCPGPGQDT, NHLRHCLSCSKCR,HCLSCSKCRKEMG, KEMGQVEISSCTV, GQVEISSCTVDRD, VEISSCTVDRDTV,CTVDRDTVCGCRK, DTVCGCRKNQYRH, NQYRHYWSENLFQ, RHYWSENLFQCFN,HYWSENLFQCFNC, ENLFQCFNCSLCL, NLFQCFNCSLCLN, QCFNCSLCLNGTV,CSLCLNGTVHLSC, LCLNGTVHLSCQE, GTVHLSCQEKQNT, VHLSCQEKQNTVC,EKQNTVCTCHAGF, NTVCTCHAGFFLR, GFFLRENECVSCS, FFLRENECVSCSN,ECVSCSNCKKSLE, KSLECTKLCLPQI, TKLCLPQIENVKG, LCLPQIENVKGTE,PQIENVKGTEDSG, SGTTVLLPLVIFF

[0204] Any of the above-cited peptide sequences can be used formodifying by exchanging one or more amino acids to obtain a sequencehaving a reduced or no immunogenicity.

EXAMPLE 11 Soluble TNF-R2

[0205] Soluble tumor necrosis factor receptor 2 (sTNF-R2) is aderivative of the human tumor necrosis factor receptor 2 describedpreviously [Smith, C. A. et al (1990) Science 248: 1019-1023; Kohno, T.et al (1990) Proc. Nat. Acad. Sci. U.S.A. 87: 8331-8335; Beltinger, C.P. et al (1996) Genomics 35:94-100] comprising the extracellular domainof the intact receptor. The soluble forms are able to bind tumournecrosis factor with high affinity and inhibit the cytotoxic activity ofthe cytokine in vitro. Recombinant preparations of sTNF-R2 are ofsignificant therapeutic value for the treatment of diseases where anexcess level of tumour necrosis factor is causing a pathogenic effect. Aparticular recombinant preparation termed ethanercept has gainedclinical approval for the treatment of rheumatoid arthritis and this andother similar agents may be of value in the treatment of otherindications such as cachexia, sepsis and autoimmune disorders.Ethanercept is a dimeric fusion protein comprising the extracellulardomain of the human TNFR2 molecule in combination with the Fc domain ofthe human IgG1 molecule. The dimeric molecule comprises 934 amino acids[U.S. Pat. No. 5,395,760; U.S. Pat. No. 5,605,690; U.S. Pat. No.5,945,397, U.S. RE36,755].

[0206] Peptide sequences in the TNF binding domain of the human TNFR2protein with potential human MHC class II binding activity are:TPYAPEPGSTCRL, CRLREYYDQTAQM, REYYDQTAQMCCS, EYYDQTAQMCCSK,AQMCCSKCSPGQH, KCSPGQHAKVFCT, AKVFCTKTSDTVC, KVFCTKTSDTVCD,STYTQLWNWVPEC, TQLWNWVPECLSC, QLWNWVPECLSCG, NWVPECLSCGSRC,ECLSCGSRCSSDQ, SRCSSDQEVTQAC, QEVTQACTREQNR, QNRICTCRPGWYC,NRICTCRPGWYCA, PGWYCALSKQEGC, GWYCALSKQEGCR, CALSKQEGCRLCA,APLRKCRPGFGVA, PGFGVARPGTETS, FGVARPGTETSDV, SDVVCKPCAPGTF,GTFSNTTSSTDIC, TDICRPHQICNVV, HQICNVVAIPGNA, ICNVVAIPGNASR,CNVVAIPGNASRD, NVVAIPGNASRDA, VAIPGNASRDAVC, DAVCTSTTTPTRS,TRSMAPGAVHLPQ, RSMAPGAVHLPQP, VHLPQPVSTRSQH, QPVSTRSQHTQPT,PEPSTAPSTSFLL, SFLLPMGPSPPAE, FLLPMGPSPPAEG

EXAMPLE 12 β-GCR

[0207] Beta-Glucocerebrosidase (b-D-glucosyl-N-acylsphingosineglucohydrolase, E.C. 3.2.1.45) is a monomeric glycoprotein of 497 aminoacid residues. The enzyme catalyses the hydrolysis of the glycolipidglucocerebroside to glucose and ceramide. Deficiency in GCR activityresults in a lysosomal storage disease referred to as Gaucher disease.The disease is characterised by the accumulation of glucocerebrosideengorged tissue macrophages that accumulate in the liver, spleen, bonemarrow and other organs. The disease has varying degrees of severityfrom type 1 disease with haematologic problems but no neuronalinvolvement, to type 2 disease manifesting early after birth withextensive neuronal involvement and is universally progressive and fatalwithin 2 years of age. Type 3 disease is also recognised in someclassifications and also shows neurologic involvement. Previously theonly useful therapy for Gaucher disease has been administration of GCRderived from human placenta (known as alglucerase) but more recentlypharmaceutical preparations of recombinant GCR (“ceredase” and“cerezyme”) have shown efficacy in the treatment of type I disease[Niederau, C. et al (1998) Eur. J. Med. Res. 3: 25-30].

[0208] Peptide sequences in human GCR with potential human MHC class IIbinding activity are: PCIPKSFGYSSVV, KSFGYSSVVCVCN, FGYSSVVCVCNAT,SSVVCVCNATYCD, SVVCVCNATYCDS, VCVCNATYCDSFD, ATYCDSFDPPTFP,DSFDPPTFPALGT, PTFPALGTFSRYE, PALGTFSRYESTR, GTFSRYESTRSGR,SRYESTRSGRRME, GRRMELSMGPIQA, RRMELSMGPIQAN, RMELSMGPIQANH,MELSMGPIQANHT, LSMGPIQANHTGT, MGPIQANHTGTGL, GPIQANHTGTGLL,TGLLLTLQPEQKF, GLLLTLQPEQKFQ, LLLTLQPEQKFQK, LTLQPEQKFQKVK,TLQPEQKFQKVKG, PEQKFQKVKGFGG, QKFQKVKGFGGAM, QKVKGFGGAMTDA,KGFGGAMTDAAAL, GFGGAMTDAAALN, GAMTDAAALNILA, AMTDAAALNILAL,MTDAAALNILALS, AALNILALSPPAQ, ALNILALSPPAQN, LNILALSPPAQNL,NILALSPPAQNLL, LALSPPAQNLLLK, ALSPPAQNLLLKS, PAQNLLLKSYFSE,AQNLLLKSYFSEE, QNLLLKSYFSEEG, NLLLKSYFSEEGI, LLLKSYFSEEGIG,KSYFSEEGIGYNI, SYFSEEGIGYNII, FSEEGIGYNIIRV, EGIGYNIIRVPMA,GIGYNIIRVPMAS, IGYNIIRVPMASC, YNIIRVPMASCDF, NIIRVPMASCDFS,IIRVPMASCDFSI, IRVPMASCDFSIR, VPMASCDFSIRTY, PMASCDFSIRTYT,SCDFSIRTYTYAD, CDFSIRTYTYADT, FSIRTYTYADTPD, RTYTYADTPDDFQ,TYTYADTPDDFQL, YTYADTPDDFQLH, ADTPDDFQLHNFS, PDDFQLHNFSLPE,DDFQLHNFSLPEE, FQLHNFSLPEEDT, HNFSLPEEDTKLK, FSLPEEDTKLKIP,SLPEEDTKLKIPL, EEDTKLKIPLIHR, TKLKIPLIHRALQ, KLKIPLIHRALQL,LKIPLIHRALQLA, IPLIHRALQLAQR, PLIHRALQLAQRP, HRALQLAQRPVSL,RALQLAQRPVSLL, ALQLAQRPVSLLA, LQLAQRPVSLLAS, RPVSLLASPWTSP,PVSLLASPWTSPT, VSLLASPWTSPTW, SLLASPWTSPTWL, SPWTSPTWLKTNG,TSPTWLKTNGAVN, PTWLKTNGAVNGK, TWLKTNGAVNGKG, GAVNGKGSLKGQP,GSLKGQPGDIYHQ, GDIYHQTWARYFV, DIYHQTWARYFVK, QTWARYFVKFLDA,WARYFVKFLDAYA, ARYFVKFLDAYAE, RYFVKFLDAYAEH, YFVKFLDAYAEHK,FVKFLDAYAEHKL, VKFLDAYAEHKLQ, KFLDAYAEHKLQF, DAYAEHKLQFWAV,YAEHKLQFWAVTA, HKLQFWAVTAENE, LQFWAVTAENEPS, QFWAVTAENEPSA,FWAVTAENEPSAG, WAVTAENEPSAGL, VTAENEPSAGLLS, PSAGLLSGYPFQC,AGLLSGYPFQCLG, GLLSGYPFQCLGF, SGYPFQCLGFTPE, YPFQCLGFTPEHQ,QCLGFTPEHQRDF, LGFTPEHQRDFIA, FTPEHQRDFIARD, RDFIARDLGPTLA,DFIARDLGPTLAN, RDLGPTLANSTHH, LGPTLANSTHHNV, PTLANSTHHNVRL,HNVRLLMLDDQRL, VRLLMLDDQRLLL, RLLMLDDQRLLLP, LLMLDDQRLLLPH,LMLDDQRLLLPHW, DDQRLLLPHWAKV, DQRLLLPHWAKVV, QRLLLPHWAKVVL,RLLLPHWAKVVLT, LLLPHWAKVVLTD, PHWAKVVLTDPEA, WAKVVLTDPEAAK,AKVVLTDPEAAKY, KVVLTDPEAAKYV, VVLTDPEAAKYVH, EAAKYVHGIAVHW,AKYVHGIAVHWYL, KYVHGIAVHWYLD, YVHGIAVHWYLDF, HGIAVHWYLDFLA,IAVHWYLDFLAPA, VHWYLDFLAPAKA, HWYLDFLAPAKAT, WYLDFLAPAKATL,LDFLAPAKATLGE, DFLAPAKATLGET, AKATLGETHRLFP, ATLGETHRLFPNT,GETHRLFPNTMLF, ETHRLFPNTMLFA, THRLFPNTMLFAS, HRLFPNTMLFASE,RLFPNTMLFASEA, FPNTMLFASEACV, NTMLFASEACVGS, TMLFASEACVGSK,MLFASEACVGSKF, ACVGSKFWEQSVR, GSKFWEQSVRLGS, SKFWEQSVRLGSW,KFWEQSVRLGSWD, QSVRLGSWDRGMQ, VRLGSWDRGMQYS, RLGSWDRGMQYSH,GSWDRGMQYSHSI, WDRGMQYSHSIIT, RGMQYSHSIITNL, MQYSHSIITNLLY,QYSHSIITNLLYH, YSHSIITNLLYHV, HSIITNLLYHVVG, SIITNLLYHVVGW,TNLLYHVVGWTDW, NLLYHVVGWTDWN, LLYHVVGWTDWNL, YHVVGWTDWNLAL,HVVGWTDWNLALN, VVGWTDWNLALNP, VGWTDWNLALNPE, TDWNLALNPEGGP,WNLALNPEGGPNW, LALNPEGGPNWVR, PNWVRNFVDSPII, NWVRNFVDSPIIV,RNFVDSPIIVDIT, NFVDSPIIVDITK, SPIIVDITKDTFY, PIIVDITKDTFYK,IIVDITKDTFYKQ, VDITKDTFYKQPM, DTFYKQPMFYHLG, TFYKQPMFYHLGH,QPMFYHLGHFSKF, PMFYHLGHFSKFI, MFYHLGHFSKFIP, YHLGHFSKFIPEG,GHFSKFIPEGSQR, SKFIPEGSQRVGL, KFIPEGSQRVGLV, IPEGSQRVGLVAS,QRVGLVASQKNDL, VGLVASQKNDLDA, GLVASQKNDLDAV, SQKNDLDAVALMH,NDLDAVALMHPDG, DAVALMHPDGSAV, VALMHPDGSAVVV, ALMHPDGSAVVVV,SAVVVVLNRSSKD, AVVVVLNRSSKDV, VVVVLNRSSKDVP, VVVLNRSSKDVPL,VVLNRSSKDVPLT, KDVPLTIKDPAVG, VPLTIKDPAVGPL, PLTIKDPAVGFLE,LTIKDPAVGFLET, PAVGFLETISPGY, VGFLETISPGYSI, GFLETISPGYSIH,FLETISPGYSIHT, ETISPGYSIHTYL, PGYSIHTYLWHRQ, PGYSIHTYLWRRQ

EXAMPLE 13 Protein C

[0209] Protein C is a vitamin K dependent serine-protease involved inthe regulation of blood coagulation. The protein is activated bythrombin to produce activated protein C which in turn degrades (downregulates) Factors Va and VIIIa in the coagulation cascade. Protein C isexpressed in the liver as a single chain precursor and undergoes aseries of processing events resulting in a molecule comprising a lightchain and a heavy chain held together by di-sulphide linkage. Protein Cis activated by cleavage of a tetradecapeptide from the N-terminus ofthe heavy chain by thrombin. Pharmaceutical preparations of protein C innative or activated form, have value in the treatment of patients withvascular disorders and or acquired deficiencies in protein C. Suchpatients include therefore individuals suffering from thrombotic stroke,or protein C deficiency associated with sepsis, transplantationprocedures, preganacy, severe burns, major surgery or other severetraumas. Protein C is also used in the treatment of individuals withhereditary protein C deficiency. This disclosure specifically pertainsthe human protein C being the mature (processed) form comprising a lightchain of 155 amino acid residues and a heavy chain of 262 amino acidresidues [Foster, D. C. et al (1985) Proc. Natl. Acad. Sci. U.S.A. 82:4673-4677; Beckman, R. J. et al (1985) Nucleic Acids Res. 13:5233-5247]. Others have provided protein C molecules including activatedprotein C formulations and methods of use [U.S. Pat. No. 6,159,468; U.S.Pat. No. 6,156,734; U.S. Pat. No. 6,037,322; U.S. Pat. No. 5,618,714].Peptide sequences in human protein C heavy-chain with potential humanMHC class II binding activity are: DQEDQVDPRLIDG, QEDQVDPRLIDGK,DQVDPRLIDGKMT, QVDPRLIDGKMTR, VDPRLIDGKMTRR, DPRLIDGKMTRRG,PRLIDGKMTRRGD, RLIDGKMTRRGDS, SPWQVVLLDSKKK, WQVVLLDSKKKLA,QVVLLDSKKKLAC, VVLLDSKKKLACG, VLLDSKKKLACGA, DSKKKLACGAVLI,SKKKLACGAVLIH, KKLACGAVLIHPS, CGAVLIHPSWVLT, GAVLIHPSWVLTA,VLIHPSWVLTAAH, PSWVLTAAHCMDE, SWVLTAAHCMDES, WVLTAAHCMDESK,AAHCMDESKKLLV, HCMDESKKLLVRL, SKKLLVRLGEYDL, KKLLVRLGEYDLR,KLLVRLGEYDLRR, LLVRLGEYDLRRW, VRLGEYDLRRWEK, RLGEYDLRRWEKW,LGEYDLRRWEKWE, GEYDLRRWEKWEL, YDLRRWEKWELDL, RRWEKWELDLDIK,EKWELDLDIKEVF, WELDLDIKEVFVH, LDLDIKEVFVHPN, LDIKEVFVHPNYS,IKEVFVHPNYSKS, KEVFVHPNYSKST, EVFVHPNYSKSTT, VFVHPNYSKSTTD,PNYSKSTTDNDIA, SKSTTDNDIALLH, TTDNDIALLHLAQ, TDNDIALLHLAQP,DNDIALLHLAQPA, NDIALLHLAQPAT, IALLHLAQPATLS, ALLHLAQPATLSQ,LHLAQPATLSQTI, AQPATLSQTIVPI, PATLSQTIVPICL, ATLSQTIVPICLP,TLSQTIVPICLPD, QTIVPICLPDSGL, TIVPICLPDSGLA, IVPICLPDSGLAE,VPICLPDSGLAER, ICLPDSGLAEREL, PDSGLAERELNQA, SGLAERELNQAGQ,GLAERELNQAGQE, LAERELNQAGQET, RELNQAGQETLVT, GQETLVTGWGYHS,ETLVTGWGYHSSR, TLVTGWGYHSSRE, TGWGYHSSREKEA, WGYHSSREKEAKR,WGYHSSREKEAKR, SREKEAKRNRTFV, RNRTFVLNFIKIP, NRTFVLNFIKIPV,RTFVLNFIKIPVV, TFVLNFIKIPVVP, FVLNFIKIPVVPH, LNFIKIPVVPHNE,NFIKIPVVPHNEC, IKIPVVPHNECSE, IPVVPHNECSEVM, PVVPHNECSEVMS,VVPHNECSEVMSN, NECSEVMSNMVSE, SEVMSNMVSENML, EVMSNMVSENMLC,VMSNMVSENMLCA, SNMVSENMLCAGI, MVSENMLCAGILG, VSENMLCAGILGD,ENMLCAGILGDRQ, NMLCAGILGDRQD, AGILGDRQDACEG, GILGDRQDACEGD,GPMVASFHGTWFL, PMVASFHGTWFLV, MVASFHGTWFLVG, ASFHGTWFLVGLV,GTWFLVGLVSWGE, TWFLVGLVSWGEG, WFLVGLVSWGEGC, FLVGLVSWGEGCG,VGLVSWGEGCGLL, GLVSWGEGCGLLH, VSWGEGCGLLHNY, EGCGLLHNYGVYT,CGLLHNYGVYTKV, GLLHNYGVYTKVS, LHNYGVYTKVSRY, HNYGVYTKVSRYL,YGVYTKVSRYLDW, GVYTKVSRYLDWI, TKVSRYLDWIHGH, SRYLDWIHGHIRD,RYLDWIHGHIRDK, LDWIHGHIRDKEA, DWIHGHIRDKEAP, HGHIRDKEAPQKS,GHIRDKEAPQKSW

[0210] Peptide sequences in human protein C light-chain with potentialhuman MHC class II binding activity are: NSFLEELRHSSLE, SFLEELRHSSLER,EELRHSSLERECI, LRHSSLERECIEE, SSLERECIEEICD, ECIEEICDFEEAK,IEEICDFEEAKEI, EEICDFEEAKEIF, EICDFEEAKEIFQ, CDFEEAKEIFQNV,KEIFQNVDDTLAF, EIFQNVDDTLAFW, IFQNVDDTLAFWS, QNVDDTLAFWSKH,DDTLAFWSKHVDG, DTLAFWSKHVDGD, LAFWSKHVDGDQC, AFWSKHVDGDQCL,WSKHVDGDQCLVL, KHVDGDQCLVLPL, QCLVLPLEHPCAS, CLVLPLEHPCASL,LVLPLEHPCASLC, LPLEHPCASLCCG, ASLCCGHGTCIDG, HGTCIDGIGSFSC,TCIDGIGSFSCDC, DGIGSFSCDCRSG, GSFSCDCRSGWEG, CRSGWEGRFCQRE,SGWEGRFCQREVS, GWEGRFCQREVSF, GRFCQREVSFLNC, RFCQREVSFLNCS,QREVSFLNCSLDN, REVSFLNCSLDNG, VSFLNCSLDNGGC, SFLNCSLDNGGCT,CSLDNGGCTHYCL, THYCLEEVGWRRC, YCLEEVGWRRCSC, EEVGWRRCSCAPG,VGWRRCSCAPGYK, RRCSCAPGYKLGD, APGYKLGDDLLQC, PGYKLGDDLLQCH,YKLGDDLLQCHPA, LGDDLLQCHPAVK, GDDLLQCHPAVKF, DDLLQCHPAVKFP,DLLQCHPAVKFPC, PAVKFPCGRPWKR, VKFPCGRPWKRME, RPWKRMEKKRSHL

EXAMPLE 14 Subtilisins

[0211] The subtilisins are a class of protease enzyme with significanteconomic and industrial importance. They may be used as components ofdetergents or cosmetics, or in the production of textiles and otherindustries and consumer preparations. Exposure of particular humansubjects to bacterial subtilisins may evoke an unwanted hypersensitivityreaction in those individuals. There is a need for subtilisin analogueswith enhanced properties and especially, improvements in the biologicalproperties of the protein. In this regard, it is highly desired toprovide subtilisins with reduced or absent potential to induce an immuneresponse in the human subject. Subtilisin proteins such as identifiedfrom other sources including bacterial, fungal or vertebrate sources,including mammalian organisms and man, have in common many of thepeptide sequences of the present disclosure and have in common manypeptide sequences with substantially the same sequence as those of thedisclosed listing. Such protein sequences equally therefore fall underthe scope of the present invention. Others have provided subtilisinmolecules including modified subtilisins [U.S. Pat. No. 5,700,676; U.S.Pat. No. 4,914,031; U.S. Pat. No. 5,397,705; U.S. Pat. No. 5,972,682].

[0212] Peptide sequences in B. lentus subtilisin with potential humanMHC class II binding activity are: QSVPWGISRVQAP, SVPWGISRVQAPA,WGISRVQAPAAHN, SRVQAPAAHNRGL, VQAPAAHNRGLTG, AHNRGLTGSGVKV,RGLTGSGVKVAVL, SGVKVAVLDTGIS, GVKVAVLDTGIST, VKVAVLDTGISTH,VAVLDTGISTHPD, AVLDTGISTHPDL, TGISTHPDLNIRG, ISTHPDLNIRGGA,HPDLNIRGGASFV, PDLNIRGGASFVP, LNIRGGASFVPGE, ASFVPGEPSTQDG,SFVPGEPSTQDGN, EPSTQDGNGHGTH, GHGTHVAGTIAAL, HGTHVAGTIAALN,THVAGTIAALNNS, AGTIAALNNSIGV, GTIAALNNSIGVL, AALNNSIGVLGVA,ALNNSIGVLGVAP, NSIGVLGVAPSAE, GVLGVAPSAELYA, LGVAPSAELYAVK,APSAELYAVKVLG, AELYAVKVLGASG, ELYAVKVLGASGS, YAVKVLGASGSGS,VKVLGASGSGSVS, KVLGASGSGSVSS, SGSGSVSSIAQGL, SGSVSSIAQGLEW,GSVSSIAQGLEWA, SSIAQGLEWAGNN, QGLEWAGNNGMHV, LEWAGNNGMHVAN,NNGMHVANLSLGS, NGMHVANLSLGSP, MHVANLSLGSPSP, HVANLSLGSPSPS,VANLSLGSPSPSA, ANLSLGSPSPSAT, LSLGSPSPSATLE, SPSPSATLEQAVN,SPSATLEQAVNSA, PSATLEQAVNSAT, ATLEQAVNSATSR, TLEQAVNSATSRG,QAVNSATSRGVLV, RGVLVVAASGNSG, GVLVVAASGNSGA, VLVVAASGNSGAG,LVVAASGNSGAGS, VAASGNSGAGSIS, GSISYPARYANAM, ISYPARYANAMAV,YPARYANAMAVGA, ARYANAMAVGATD, NAMAVGATDQNNN, MAVGATDQNNNRA,AVGATDQNNNRAS, NNRASFSQYGAGL, RASFSQYGAGLDI, ASFSQYGAGLDIV,SQYGAGLDIVAPG, GAGLDIVAPGVNV, AGLDIVAPGVNVQ, LDIVAPGVNVQST,DIVAPGVNVQSTY, APGVNVQSTYPGS, PGVNVQSTYPGST, VNVQSTYPGSTYA,STYPGSTYASLNG, STYASLNGTSMAT, ASLNGTSMATPHV, NGTSMATPHVAGA,MATPHVAGAAALV, TSMATPHVAGAAA, PHVAGAAALVKQK, AALVKQKNPSWSN,ALVKQKNPSWSNV, PSWSNVQIRNHLK, WSNVQIRNHLKNT, SNVQIRNHLKNTA,VQIRNHLKNTATS, QIRNHLKNTATSL, RNHLKNTATSLGS, NHLKNTATSLGST,HLKNTATSLGSTN, ATSLGSTNLYGSG, TSLGSTNLYGSGL, LGSTNLYGSGLVN,TNLYGSGLVNAEA, NLYGSGLVNAEAA, LYGSGLVNAEAAT,

[0213] Peptide sequences in B. amyloliquefaciens subtilisin withpotential human MHC class II binding activity are: QSVPYGVSQIKAP,SVPYGVSQIKAPA, VPYGVSQIKAPAL, YGVSQIKAPALHS, VSQIKAPALHSQG,SQIKAPALHSQGY, PALHSQGYTGSNV, QGYTGSNVKVAVI, SNVKVAVIDSGID,VKVAVIDSGIDSS, KVAVIDSGIDSSH, VAVIDSGIDSSHP, AVIDSGIDSSHPD,VIDSGIDSSHPDL, SGIDSSHPDLKVA, DSSHPDLKVAGGA, SHPDLKVAGGASM,HPDLKVAGGASMV, PDLKVAGGASMVP, DLKVAGGASMVPS, LKVAGGASMVPSE,GGASMVPSETNPF, ASMVPSETNPFQD, SMVPSETNPFQDN, NPFQDNNSHGTHV,FQDNNSHGTHVAG, SHGTHVAGTVAAL, HGTHVAGTVAALN, THVAGTVAALNNS,AGTVAALNNSIGV, GTVAALNNSIGVL, AALNNSIGVLGVA, ALNNSIGVLGVAP,NNSIGVLGVAPSA, NSIGVLGVAPSAS, SIGVLGVAPSASL, IGVLGVAPSASLY,GVLGVAPSASLYA, LGVAPSASLYAVK, APSASLYAVKVLG, ASLYAVKVLGADG,SLYAVKVLGADGS, YAVKVLGADGSGQ, VKVLGADGSGQYS, KVLGADGSGQYSW,ADGSGQYSWIING, GQYSWIINGIEWA, YSWIINGIEWAIA, SWIINGIEWAIAN,WIINGIEWAIANN, NGIEWAIANNMDV, IEWAIANNMDVIN, WAIANNMDVINMS,ANNMDVINMSLGG, NNMDVINMSLGGP, MDVINMSLGGPSG, DVINMSLGGPSGS,INMSLGGPSGSAA, MSLGGPSGSAALK, AALKAAVDKAVAS, ALKAAVDKAVASG,AAVDKAVASGVVV, AVDKAVASGVVVV, KAVASGVVVVAAA, SGVVVVAAAGNEG,GVVVVAAAGNEGT, VVVVAAAGNEGTS, VVVAAAGNEGTSG, AAAGNEGTSGSSS,SSTVGYPGKYPSV, STVGYPGKYPSVI, VGYPGKYPSVIAV, GKYPSVIAVGAVD,PSVIAVGAVDSSN, SVIAVGAVDSSNQ, IAVGAVDSSNQRA, GAVDSSNQRASFS,VDSSNQRASFSSV, ASFSSVGPELDVM, SSVGPELDVMAPG, GPELDVMAPGVSI,PELDVMAPGVSIQ, ELDVMAPGVSIQS, LDVMAPGVSIQST, DVMAPGVSIQSTL,APGVSIQSTLPGN, PGVSIQSTLPGNK, VSIQSTLPGNKYG, STLPGNKYGAYNG,GNKYGAYNGTSMA, NKYGAYNGTSMAS, GAYNGTSMASPHV, YNGTSMASPHVAG,TSMASPHVAGAAA, MASPHVAGAAALI, PHVAGAAALILSK, AAALILSKHPNWT,AALILSKHPNWTN, ALILSKHPNWTNT, LILSKHPNWTNTQ, PNWTNTQVRSSLE,TQVRSSLENTTTK, QVRSSLENTTTKL, VRSSLENTTTKLG, SSLENTTTKLGDS,TKLGDSFYYGKGL, LGDSFYYGKGLIN, DSFYYGKGLINVQ, SFYYGKGLINVQA,FYYGKGLINVQAA, YYGKGLINVQAAA

[0214] Peptide sequences in B.subtilis subtilisin with potential humanMHC class II binding activity are: QSVPYGISQIKAP, SVPYGISQIKAPA,VPYGISQIKAPAL, YGISQIKAPALHS, ISQIKAPALHSQG, SQIKAPALHSQGY,PALHSQGYTGSNV, QGYTGSNVKVAVI, SNVKVAVIDSGID, VKVAVIDSGIDSS,KVAVIDSGIDSSH, VAVIDSGIDSSHP, AVIDSGIDSSHPD, VIDSGIDSSHPDL,SGIDSSHPDLNVR, DSSHPDLNVRGGA, HPDLNVRGGASFV, PDLNVRGGASFVP,DLNVRGGASFVPS, LNVRGGASFVPSE, GGASFVPSETNPY, ASFVPSETNPYQD,SFVPSETNPYQDG, NPYQDGGSHGTHV, SHGTHVAGTIAAL, HGTHVAGTIAALN,THVAGTIAALNNS, AGTIAALNNSIGV, GTIAALNNSIGVL, AALNNSIGVLGVS,ALNNSIGVLGVSP, NNSIGVLGVSPSA, NSIGVLGVSPSAS, IGVLGVSPSASLY,GVLGVSPSASLYA, LGVSPSASLYAVK, SPSASLYAVKVLD, ASLYAVKVLDSTG,YAVKVLDSTGSGQ, VKVLDSTGSGQYS, KVLDSTGSGQYSW, STGSGQYSWIING,GQYSWIINGIEWA, YSWIINGIEWAIS, SWIINGIEWAISN, WIINGIEWAISNN,NGIEWAISNNMDV, IEWAISNNMDVIN, WAISNNMDVINMS, SNNMDVINMSLGG,NNMDVINMSLGGP, MDVINMSLGGPTG, DVINMSLGGPTGS, INMSLGGPTGSTA,MSLGGPTGSTALK, TALKTVVDKAVSS, ALKTVVDKAVSSG, KTVVDKAVSSGIV,TVVDKAVSSGIVV, VVDKAVSSGIVVA, KAVSSGIVVAAAA, VSSGIVVAAAAGN,SGIVVAAAAGNEG, GIVVAAAAGNEGS, IVVAAAAGNEGSS, VVAAAAGNEGSSG,AAAGNEGSSGSTS, TSTVGYPAKYPST, STVGYPAKYPSTI, VGYPAKYPSTIAV,AKYPSTIAVGAVN, PSTIAVGAVNSSN, STIAVGAVNSSNQ, TIAVGAVNSSNQR,IAVGAVNSSNQRA, GAVNSSNQRASFS, VNSSNQRASFSSA, NQRASFSSAGSEL,ASFSSAGSELDVM, GSELDVMAPGVSI, ELDVMAPGVSIQS, SELDVMAPGVSIQ,LDVMAPGVSIQST, DVMAPGVSIQSTL, APGVSIQSTLPGG, PGVSIQSTLPGGT,VSIQSTLPGGTYG, STLPGGTYGAYNG, GGTYGAYNGTSMA, GTYGAYNGTSMAT,GAYNGTSMATPHV, YNGTSMATPHVAG, TSMATPHVAGAAA, MATPHVAGAAALI,PHVAGAAALILSK, GAAALILSKHPTW, AALILSKHPTWTN, ALILSKHPTWTNA,LILSKHPTWTNAQ, PTWTNAQVRDRLE, AQVRDRLESTATY, QVRDRLESTATYL,DRLESTATYLGNS, ATYLGNSFYYGKG, TYLGNSFYYGKGL, LGNSFYYGKGLIN,NSFYYGKGLINVQ, SFYYGKGLINVQA, FYYGKGLINVQAA, YYGKGLINVQAAA

[0215] Peptide sequences in B. licheniformis subtilisin with potentialhuman MHC class II binding activity are: QTVPYGIPLIKAD, VPYGIPLIKADKV,YGIPLIKADKVQA, IPLIKADKVQAQG, PLIKADKVQAQGF, IKADKVQAQGFKG,DKVQAQGFKGANV, QGFKGANVKVAVL, ANVKVAVLDTGIQ, VKVAVLDTGIQAS,KVAVLDTGIQASH, VAVLDTGIQASHP, AVLDTGIQASHPD, VLDTGIQASHPDL,DTGIQASHPDLNV, TGIQASHPDLNVV, QASHPDLNVVGGA, HPDLNVVGGASFV,PDLNVVGGASFVA, DLNVVGGASFVAG, LNVVGGASFVAGE, NVVGGASFVAGEA,ASFVAGEAYNTDG, SFVAGEAYNTDGN, EAYNTDGNGHGTH, GHGTHVAGTVAAL,HGTHVAGTVAALD, THVAGTVAALDNT, GTVAALDNTTGVL, TVAALDNTTGVLG,AALDNTTGVLGVA, DNTTGVLGVAPSV, TTGVLGVAPSVSL, TGVLGVAPSVSLY,GVLGVAPSVSLYA, LGVAPSVSLYAVK, APSVSLYAVKVLN, PSVSLYAVKVLNS,VSLYAVKVLNSSG, SLYAVKVLNSSGS, YAVKVLNSSGSGS, VKVLNSSGSGSYS,KVLNSSGSGSYSG, GSYSGIVSGIEWA, TNGMDVINMSLGG, NGMDVINMSLGGA,MDVINMSLGGASG, DVINMSLGGASGS, INMSLGGASGSTA, MSLGGASGSTAMK,TAMKQAVDNAYAR, AMKQAVDNAYARG, QAVDNAYARGVVV, NAYARGVVVVAAA,RGVVVVAAAGNSG, GVVVVAAAGNSGN, VVVVAAAGNSGNS, VVVAAAGNSGNSG,NTIGYPAKYDSVI, IGYPAKYDSVIAV, AKYDSVIAVGAVD, DSVIAVGAVDSNS,SVIAVGAVDSNSN, IAVGAVDSNSNRA, AVGAVDSNSNRAS, GAVDSNSNRASFS,AVDSNSNRASFSS, SNRASFSSVGAEL, ASFSSVGAELEVM, SSVGAELEVMAPG,GAELEVMAPGAGV, AELEVMAPGAGVY, ELEVMAPGAGVYS, LEVMAPGAGVYST,EVMAPGAGVYSTY, APGAGVYSTYPTN, AGVYSTYPTNTYA, GVYSTYPTNTYAT,STYPTNTYATLNG, NTYATLNGTSMAS, ATLNGTSMASPHV, LNGTSMASPHVAG,TSMASPHVAGAAA, MASPHVAGAAALI, PHVAGAAALILSK, GAAALILSKHPNL,AALILSKHPNLSA, ALILSKHPNLSAS, LILSKHPNLSASQ, SKHPNLSASQVRN,HPNLSASQVRNRL, PNLSASQVRNRLS, LSASQVRNRLSST, SQVRNRLSSTATY,QVRNRLSSTATYL, NRLSSTATYLGSS, ATYLGSSFYYGKG, TYLGSSFYYGKGL,LGSSFYYGKGLIN, SSFYYGKGLINVE, SFYYGKGLINVEA, FYYGKGLINVEAA,YYGKGLINVEAAA

EXAMPLE 15 Ligands of CNTF

[0216] The present invention provides for modified forms of the proteinsubunits comprising a heterodimeric ligand for the ciliary neurotrophicfactor (CNTF) receptor complex in humans. The receptor complex isactivated by at least two ligands including CNTF and a heterodimericcomplex comprising cardiotrophin-like cytokine (CLC) and the solublereceptor cytokine-like factor 1 (CLF) [Elson G. C. A. et al (2000)Nature Neuroscience 3: 867-872]. CLC is a protein of the IL-6 family ofcytokines and is also known as novel neurotrophin-1/B cell-stimulatingfactor-3 [Senaldi, G. et al (1999) Proc. Nat. Acad. Sci. USA 96:11458-11463, U.S. Pat. No. 5,741,772]. CLF is homologous to proteins ofthe cytokine type I receptor family [Elson, G. C. A. et al (1998)Journal of Immunol. 161: 1371-1379] and has also been identified as NR6[Alexander W. S. et al (1999) Curr. Biol. 9: 605-608]. Heterodimersformed by association of CLC and CLF have been shown to directlyinteract with the CNTFR and the so formed trimeric complex is able tostimulate signalling events within cells expressing the other recognisedcomponents of the CNTFR complex such as gp130 and LIPR [Elson G. C. A.et al (2000) ibid].

[0217] Peptide sequences in human CLC with potential human MHC class IIbinding activity are: PGPSIQKTYDLTR, PSIQKTYDLTRYL, IQKTYDLTRYLEH,KTYDLTRYLEHQL, YDLTRYLEHQLRS, LTRYLEHQLRSLA, TRYLEHQLRSLAG,RYLEHQLRSLAGT, HQLRSLAGTYLNY, QLRSLAGTYLNYL, RSLAGTYLNYLGP,GTYLNYLGPPFNE, TYLNYLGPPFNEP, NYLGPPFNEPDFN, PPFNEPDFNPPRL,PFNEPDFNPPRLG, PDFNPPRLGAETL, FNPPRLGAETLPR, PRLGAETLPRATV,LGAETLPRATVDL, ETLPRATVDLEVW, PRATVDLEVWRSL, ATVDLEVWRSLND,TVDLEVWRSLNDK, VDLEVWRSLNDKL, LEVWRSLNDKLRL, EVWRSLNDKLRLT,VWRSLNDKLRLTQ, RSLNDKLRLTQNY, DKLRLTQNYEAYS, KLRLTQNYEAYSH,LRLTQNYEAYSHL, TQNYEAYSHLLCY, QNYEAYSHLLCYL, EAYSHLLCYLRGL,SHLLCYLRGLNRQ, HLLCYLRGLNRQA, LCYLRGLNRQAAT, CYLRGLNRQAATA,RGLNRQAATAELR, GLNRQAATAELRR, QAATAELRRSLAH, AATAELRRSLAHF,AELRRSLAHFCTS, ELRRSLAHFCTSL, RSLAHFCTSLQGL, AHFCTSLQGLLGS,TSLQGLLGSIAGV, SLQGLLGSIAGVM, QGLLGSIAGVMAA, GLLGSIAGVMAAL,LLGSIAGVMAALG, GSIAGVMAALGYP, SIAGVMAALGYPL, AGVMAALGYPLPQ,GVMAALGYPLPQP, AALGYPLPQPLPG, LGYPLPQPLPGTE, YPLPQPLPGTEPT,QPLPGTEPTWTPG, PTWTPGPAHSDFL, WTPGPAHSDFLQK, HSDFLQKMDDFWL,SDFLQKMDDFWLL, DFLQKMDDFWLLK, FLQKMDDFWLLKE, QKMDDFWLLKELQ,DDFWLLKELQTWL, DFWLLKELQTWLW, FWLLKELQTWLWR, WLLKELQTWLWRS,KELQTWLWRSAKD, ELQTWLWRSAKDF, QTWLWRSAKDFNR, TWLWRSAKDFNRL,WLWRSAKDFNRLK, WRSAKDFNRLKKK, RSAKDFNRLKKKM, KDFNRLKKKMQPP,NRLKKKMQPPAAA, RLKKKMQPPAAAV, LKKKMQPPAAAVT, KKMQPPAAAVTLH,KMQPPAAAVTLHL, QPPAAAVTLHLGA

[0218] Peptide sequences in human CLF with potential human MHC class IIbinding activity are: TAVISPQDPTLLI, AVISPQDPTLLIG, VISPQDPTLLIGS,QDPTLLIGSSLLA, DPTLLIGSSLLAT, PTLLIGSSLLATC, TLLIGSSLLATCS,LLIGSSLLATCSV, IGSSLLATCSVHG, SSLLATCSVHGDP, SLLATCSVHGDPP,CSVHGDPPGATAE, GDPPGATAEGLYW, EGLYWTLNGRRLP, GLYWTLNGRRLPP,WTLNGRRLPPELS, RRLPPELSRVLNA, RLPPELSRVLNAS, PELSRVLNASTLA,ELSRVLNASTLAL, LSRVLNASTLALA, SRVLNASTLALAL, RVLNASTLALALA,LNASTLALALANL, NASTLALALANLN, STLALALANLNGS, LALALANLNGSRQ,LALANLNGSRQRS, ANLNGSRQRSGDN, DNLVCHARDGSIL, NLVCHARDGSILA,VCHARDGSILAGS, RDGSILAGSCLYV, DGSILAGSCLYVG, GSILAGSCLYVGL,SILAGSCLYCGLP, SCLYVGLPPEKPV, CLYVGLPPEKPVN, LYVGLPPEKPVNI,VGLPPEKPVNISC, KPVNISCWSKNMK, VNISCWSKNMKDL, KNMKDLTCRWTPG,KDLTCRWTPGAHG, CRWTPGAHGETFL, RWTPGAHGETFLH, HGETFLHTNYSLK,ETFLHTNYSLKYK, TFLHTNYSLKYKL, TNYSLKYKLRWYG, YSLKYKLRWYGQD,LKYKLRWYGQDNT, YKLRWYGQDNTCE, LRWYGQDNTCEEY, RWYGQDNTCEEYH,EEYHTVGPHSCHI, HTVGPHSCHIPKD, PHSCHIPKDLALF, CHIPKDLALFTPY,IPKDLALFTPYEI, KDLALFTPYEIWV, ALFTPYEIWVEAT, TPYEIWVEATNRL,YEIWVEATNRLGS, EIWVEATNRLGSA, IWVEATNRLGSAR, EIWVEATNRLGSA,NRLGSARSDVLTL, EATNRLGSARSDV, SARSDVLTLDILD, SDVLTLDILDVVT,DVLTLDILDVVTT, LTLDILDVVTTDP, LDILDVVTTDPPP, DILDVVTTDPPPD,LDVVTTDPPPDVH, ARSDVLTLDILDV, PDVHVSRVGGLED, VHVSRVGGLEDQL,SRVGGLEDQLSVR, DVVTTDPPPDVHV, GGLEDQLSVRWVS, RVGGLEDQLSVRW,DQLSVRWVSPPAL, LSVRWVSPPALKD, VRWVSPPALKDFL, RWVSPPALKDFLF,GLEDQLSVRWVSP, PALKDFLFQAKYQ, KDFLFQAKYQIRY, DFLFQAKYQIRYR,FLFQAKYQIRYRV, VSPPALKDFLFQA, AKYQIRYRVEDSV, YQIRYRVEDSVDW,IRYRVEDSVDWKV, YRVEDSVDWKVVD, FQAKYQIRYRVED, DSVDWKVVDDVSN,VDWKVVDDVSNQT, VEDSVDWKVVDDV, KVVDDVSNQTSCR, DDVSNQTSCRLAG,WKVVDDVSNQTSC, QTSCRLAGLKPGT, CRLAGLKPGTVYF, AGLKPGTVYFVQV,GTVYFVQVRCNPF, TVYFVQVRCNPFG, VYFVQVRCNPFGI, FVQVRCNPFGIYG,VQVRCNPFGIYGS, NPFGIYGSKKAGI, PFGIYGSKKAGIW, FGIYGSKKAGIWS,GIYGSKKAGIWSE, SKKAGIWSEWSHP, AGIWSEWSHPTAA, GIWSEWSHPTAAS,SEWSHPTAASTPR, SHPTAASTPRSER, PSSGPVRRELKQF, GPVRRELKQFLGW,RELKQFLGWLKKH, KQFLGWLKKHAYC, QFLGWLKKHAYCS, LGWLKKHAYCSNL,GWLKKHAYCSNLS, HAYCSNLSFRLYD, AYCSNLSFRLYDQ, SNLSFRLYDQWRA,LSFRLYDQWRAWM, FRLYDQWRAWMQK, RLYDQWRAWMQKS, DQWRAWMQKSHKT,RAWMQKSHKTRNQ, AWMQKSHKTRNQD, HKTRNQDEGILPS, EGILPSGRRGTAR,GILPSGRRGTARG,

EXAMPLE 16 Follicle-Stimulating Hormone

[0219] The present invention provides for modified forms of human hFSHwith one or more T cell epitopes removed. hFSH is a glycoprotein hormonewith a dimeric structure containing two glycoprotein subunits. Theprotein is being used therapeutically in the treatment of humaninfertility and a recombinant form of the protein has been the subjectof a number of clinical trials [Out, H. J. et al (1995) Hum. Reprod. 10:2534-2540; Hedon, B. et al (1995) Hum. Reprod. 10: 3102-3106;Recombinant Human FSH study Group (1995) Fertil. Steril. 63:77-86;Prevost, R. R. (1998) Pharmacotherapy 18: 1001-1010].

[0220] Peptide sequences in human hFSH with potential human MHC class IIbinding activity are: KTLQFFFLFCCWK, LQFFFLFCCWKAI, QFFFLFCCWKAIC,FFFLFCCWKAICC, FFLFCCWKAICCN, FLFCCWKAICCNS, CCWKAICCNSCEL,KAICCNSCELTNI, CELTNITIAIEKE, TNITIAIEKEECR, ITIAIEKEECRFC,IAIEKEECRFCIS, CRFCISINTTWCA, FCISINTTWCAGY, ISINTTWCAGYCY,TTWCAGYCYTRDL, AGYCYTRDLVYKD, YCYTRDLVYKDPA, RDLVYKDPARPKI,DLVYKDPARPKIQ, LVYKDPARPKIQK, PKIQKTCTFKELV, CTFKELVYETVRV,KELVYETVRVPGC, ELVYETVRVPGCA, LVYETVRVPGCAH, ETVRVPGCAHHAD,VRVPGCAHHADSL, DSLYTYPVATQCH, SLYTYPVATQCHC, YTYPVATQCHCGK,YPVATQCHCGKCD, CTVRGLGPSYCSF, RGLGPSYCSFGEM

EXAMPLE 16 Ricin A

[0221] The present invention provides for modified forms of ricin toxinA-chain (RTA) with one or more T cell epitopes removed. Ricin is acytotoxin originally isolated from the seeds of the castor plant and isan example of a type II ribosome inactivating protein (RIP). The nativemature protein is a heterodimer comprising the RTA of 267 amino acidresidues in disulphide linkage with the ricin B-chain of 262 amino acidresidues. The B-chain is a lectin with binding affinity forgalactosides. The native protein is able to bind cells via the B-chainand enters the cell by endocytosis. Inside the cell, the RTA is releasedfrom the B-chain by reduction of the disulphide linkage and is releasedfrom the endosome into the cytoplasm via unknown mechanisms. In thecytoplasm the toxin degrades ribosomes by action as a specificN-glycosylase rapidly resulting in the cessation of protein translationand cell death. The extreme cytotoxicity of RTA and other RIPs has leadto their use in experimental therapies for the treatment of cancer andother diseases where ablation of a particular cell population isrequired. Immunotoxin molecules containing antibody molecules in linkagewith RTA have been produced and used in a number of clinical trials[Ghetie, M. A. et al (1991) Cancer Res. 51: 5876-5880; Vitetta, E. S. etal (1991) Cancer Res. 51: 4052-4058; Amlot, P. L. et al (1993) Blood 82:2624-2633; Conry, R. M. et al (1995) J. Immunother. Emphasis TumorImmunol. 18: 231-241; Schnell, R. et al (2000) Leukaemia 14: 129-135].In the immunotoxin the antibody domain provides binding to the surfaceof the desired target cell and linkage to the RTA may be via chemicalcross-linkage or as a recombinant fusion protein.

[0222] Peptide sequences in ricin toxin a-chain with potential human MHCclass II binding activity are: KQYPIINFTTAGA, YPIINFTTAGATV,PIINFTTAGATVQ, INFTTAGATVQSY, ATVQSYTNFIRAV, QSYTNFIRAVRGR,TNFIRAVRGRLTT, NFIRAVRGRLTTG, RAVRGRLTTGADV, GRLTTGADVRHEI,ADVRHEIPVLPNR, HEIPVLPNRVGLP, IPVLPNRVGLPIN, PVLPNRVGLPINQ,NRVGLPINQRFIL, VGLPINQRFILVE, LPINQRFILVELS, QRFILVELSNHAE,RFILVELSNHAEL, FILVELSNHAELS, ILVELSNHAELSV, VELSNHAELSVTL,AELSVTLALDVTN, LSVTLALDVTNAY, VTLALDVTNAYVV, LALDVTNAYVVGY,LDVTNAYVVGYRA, NAYVVGYRAGNSA, AYVVGYRAGNSAY, YVVGYRAGNSAYF,VGYRAGNSAYFFH, SAYFFHPDNQEDA, AYFFHPDNQEDAE, YFFHPDNQEDAEA,EAITHLFTDVQNR, THLFTDVQNRYTF, HLFTDVQNRYTFA, TDVQNRYTFAFGG,NRYTFAFGGNYDR, YTFAFGGNYDRLE, FAFGGNYDRLEQL, GNYDRLEQLAGNL,DRLEQLAGNLREN, EQLAGNLRENIEL, GNLRENIELGNGP, ENIELGNGPLEEA,IELGNGPLEEAIS, GPLEEAISALYYY, EAISALYYYSTGG, SALYYYSTGGTQL,ALYYYSTGGTQLP, LYYYSTGGTQLPT, YYYSTGGTQLPTL, TQLPTLARSFIIC,PTLARSFIICIQM, RSFIICIQMISEA, SFIICIQMISEAA, FIICIQMISEAAR,ICIQMISEAARFQ, IQMISEAARFQYI, QMISEAARFQYIE, ARFQYIEGEMRTR,FQYIEGEMRTRIR, QYIEGEMRTRIRY, GEMRTRIRYNRRS, TRIRYNRRSAPDP,IRYNRRSAPDPSV, PSVITLENSWGRL, SVITLENSWGRLS, ITLENSWGRLSTA,NSWGRLSTAIQES, GRLSTAIQESNQG, TAIQESNQGAFAS, GAFASPIQLQRRN,SPIQLQRRNGSKF, IQLQRRNGSKFSV, SKFSVYDVSILIP, FSVYDVSILIPII,SVYDVSILIPIIA, YDVSILIPIIALM, VSILIPIIALMVY, SILIPIIALMVYR,IPIIALMVYRCAP, IALMVYRCAPPPS, ALMVYRCAPPPSS, LMVYRCAPPPSSQ,MVYRCAPPPSSQF

EXAMPLE 17 Adipocyte Complement-Related Protein

[0223] The present invention provides for modified forms of human ormouse Acrp30 with one or more T cell epitopes removed. Acrp30 is anabundant serum protein of approximately 30 kDa molecular weightexpressed exclusively by adipocyte cells [Scherer, P. E. et al (1995) J.Biol. Chem. 270: 26746-26749]. The human gene Acrp30 protein sequence isdisclosed e.g. in U.S. Pat. No. 5,869,330. Secretion of the protein isenhanced by insulin and levels of the protein are decreased in obesesubjects. The protein is involved in the regulation of energy balanceand in particular the regulation of fatty acid metabolism. Four sequencedomains are identified in the mouse and human protein comprising acleaved N-terminal signal, a region with no recognized homology to otherproteins, a collagen-like domain and a globular domain. The globulardomain may be removed from the mouse protein by protease treatment toproduce gAcrp30. Preparations of murine gAcrp30 have pharmaceuticalproperties and have been shown to decrease elevated levels of free fattyacids in the serum of mice following administration of high fat meals ori.v. injection of lipid [Fruebis, J. et al (2001) Proc. Natl. Acad. Sci.U.S.A. 98: 2005-2010].

[0224] Peptide sequences in mouse Acrp30 with potential human MHC classII binding activity are: DDVTTTEELAPAL, TTTEELAPALVPP, EELAPALVPPPKG,LAPALVPPPKGTC, PALVPPPKGTCAG, ALVPPPKGTCAGW, AGWMAGIPGHPGH,GWMAGIPGHPGHN, AGIPGHPGHNGTP, GTPGRDGRDGTPG, GDAGLLGPKGETG,AGLLGPKGETGDV, GLLGPKGETGDVG, GETGDVGMTGAEG, GDVGMTGAEGPRG,VGMTGAEGPRGFP, RGFPGTPGRKGEP, TPGRKGEPGEAAY, GRKGEPGEAAYMY,AAYMYRSAFSVGL, AYMYRSAFSVGLE, YMYRSAFSVGLET, SAFSVGLETRVTV,FSVGLETRVTVPN, VGLETRVTVPNVP, GLETRVTVPNVPI, ETRVTVPNVPIRF,TRVTVPNVPIRFT, VTVPNVPIRFTKI, VPNVPIRFTKIFY, PNVPIRFTKIFYN,VPIRFTKIFYNQQ, IRFTKIFYNQQNH, RFTKIFYNQQNHY, TKIFYNQQNHYDG,KIFYNQQNHYDGS, IFYNQQNHYDGST, QQNHYDGSTGKFY, NHYDGSTGKFYCN,GKFYCNIPGLYYF, KFYCNIPGLYYFS, CNIPGLYYFSYHI, PGLYYFSYHITVY,GLYYFSYHITVYM, LYYFSYHITVYMK, YYFSYHITVYMKD, FSYHITVYMKDVK,SYHITVYMKDVKV, YHITVYMKDVKVS, HITVYMKDVKVSL, ITVYMKDVKVSLF,TVYMKDVKVSLFK, VYMKDVKVSLFKK, KDVKVSLFKKDKA, VKVSLFKKDKAVL,VSLFKKDKAVLFT, SLFKKDKAVLFTY, FKKDKAVLFTYDQ, KDKAVLFTYDQYQ,KAVLFTYDQYQEK, AVLFTYDQYQEKN, VLFTYDQYQEKNV, FTYDQYQEKNVDQ,YDQYQEKNVDQAS, DQYQEKNVDQASG, EKNVDQASGSVLL, KNVDQASGSVLLH,ASGSVLLHLEVGD, GSVLLHLEVGDQV, SVLLHLEVGDQVW, VLLHLEVGDQVWL,LHLEVGDQVWLQV, LEVGDQVWLQVYG, DQVWLQVYGDGDH, QVWLQVYGDGDHN,VWLQVYGDGDHNG, LQVYGDGDHNGLY, QVYGDGDHNGLYA, VYGDGDHNGLYAD,GDHNGLYADNVND, NGLYADNVNDSTF, GLYADNVNDSTFT, LYADNVNDSTFTG,DNVNDSTFTGFLL, VNDSTFTGFLLYH, STFTGFLLYHDTN

[0225] Peptide sequences in human Acrp30 with potential human MHC classII binding activity are: PGVLLPLPKGACT, GVLLPLPKGACTG, VLLPLPKGACTGW,LPLPKGACTGWNA, PLPKGACTGWMAG, TGWMAGIPGHPGH, GWMAGIPGHPGHN,AGIPGHPGHNGAP, GAPGRDGRDGTPG, GDPGLIGPKGDIG, PGLIGPKGDIGET,GLIGPKGDIGETG, GPKGDIGETGVPG, GDIGETGVPGAEG, TGVPGAEGPRGFP,RGFPGIQGRKGEP, PGIQGRKGEPGEG, GRKGEPGEGAYVY, GAYVYRSAFSVGL,AYVYRSAFSVGLE, YVYRSAFSVGLET, RSAFSVGLETYVT, SAFSVGLETYVTI,AFSVGLETYVTIP, FSVGLETYVTIPN, VGLETYVTIPNMP, GLETYVTIPNMPI,ETYVTIPNMPIRF, TYVTIPNMPIRFT, VTIPNMPIRFTKI, IPNMPIRFTKIFY,PNMPIRFTKIFYN, MPIRFTKIFYNQQ, IRFTKIFYNQQNH, RFTKIFYNQQNHY,TKIFYNQQNHYDG, KIFYNQQNHYDGS, IFYNQQNHYDGST, QQNHYDGSTGKFH,NHYDGSTGKFHCN, GKFHCNIPGLYYF, CNIPGLYYFAYHI, PGLYYFAYHITVY,GLYYFAYHITVYM, LYYFAYHITVYMK, YYFAYHITVYMKD, FAYHITVYMKDVK,AYHITVYMKDVKV, YHITVYMKDVKVS, HITVYMKDVKVSL, ITVYMKDVKVSLF,TVYMKDVKVSLFK, VYMKDVKVSLFKK, KDVKVSLFKKDKA, VKVSLFKKDKAML,VSLFKKDKAMLFT, SLFKKDKAMLFTY, FKKDKAMLFTYDQ, KDKAMLFTYDQYQ,KAMLFTYDQYQEN, AMLFTYDQYQENN, MLFTYDQYQENNV, FTYDQYQENNVDQ,YDQYQENNVDQAS, DQYQENNVDQASG, ENNVDQASGSVLL, NNVDQASGSVLLH,ASGSVLLHLEVGD, GSVLLHLEVGDQV, SVLLHLEVGDQVW, VLLHLEVGDQVWL,LHLEVGDQVWLQV, LEVGDQVWLQVYG, DQVWLQVYGEGER, QVWLQVYGEGERN,VWLQVYGEGERNG, LQVYGEGERNGLY, QVYGEGERNGLYA, NGLYADNDNDSTF,GLYADNDNDSTFT, LYADNDNDSTFTG, DNDSTFTGFLLYH, STFTGFLLYHDTN.

EXAMPLE 18 Anti-C5 Antibody

[0226] The present invention provides for modified forms of monoclonalantibodies with binding specificity directed to the human C5 complementprotein. The invention provides for modified antibodies with one or moreT cell epitopes removed. The antibodies with binding specificity to C5complement protein block cleavage activation of the C5 convertase andthereby inhibit the production of the pro-inflammatory components C5aand C5b-9. Activation of the complement system is a significantcontributory factor in the pathogenesis of a number of acute and chronicdiseases, and inhibition of the complement cascade at the level of C5offers significant promise as a therapeutic avenue for some of these[Morgan B. P. (1994) Eur. J. Clin. Invest. 24: 219-228]. A number ofanti-C5 antibodies and methods for their therapeutic use have beendescribed in the art [Wurzner R. et al (1991) Complement Inflamm. 8:328-340; Thomas, T. C. et al (1996) Molecular Immunology 33: 1389-14012;U.S. Pat. No. 5,853,722; U.S. Pat. No. 6,074,64]. The antibodydesignated 5G1.1 [Thomas, T. C. et al (1996) ibid] and a single-chainhumanised variant are undergoing clinical trials for a number of diseaseindications including cardiopulmonary bypass [Fitch, J. C. K. et al(1999) Circulation 100: 2499-2506] and rheumatoid arthritis. Theinvention discloses sequences identified within the anti-C5 antibodydesignated 5G1.1 [Thomas, T. C. et al]. The sequences disclosed arederived from the variable region domains of both the heavy and lightchains of the antibody sequence that are potential T cell epitopes byvirtue of MHC class II binding potential. The disclosure furtheridentifies potential epitopes within the protein sequence of asingle-chain and “humanised” variant 5G1.1 antibody [Thomas, T. C. et al(1996) ibid].

[0227] Peptide sequences in the heavy-chain variable region of antibody5G1.1 with potential human MHC class II binding activity are:VQLQQSGAELMKP, QSGAELMKPGASV, AELMKPGASVKMS, ELMKPGASVKMSC,ASVKMSCKATGYI, VKMSCKATGYIFS, KMSCKATGYIFSN, ATGYIFSNYWIQW,TGYIFSNYWIQWI, GYIFSNYWIQWIK, YIFSNYWIQWIKQ, SNYWIQWIKQRPG,NYWIQWIKQRPGH, YWIQWIKQRPGHG, IQWIKQRPGHGLE, QWIKQRPGHGLEW,HGLEWIGEILPGS, LEWIGEILPGSGS, EWIGEILPGSGST, WIGEILPGSGSTE,GEILPGSGSTEYT, EILPGSGSTEYTE, TEYTENFKDKAAF, EWFKDKAAFTADT,FKDKAAFTADTSS, KAAFTADTSSNTA, AAFTADTSSNTAY, TAYMQLSSLTSED,AYMQLSSLTSEDS, MQLSSLTSEDSAV, SSLTSEDSAVYYC, SLTSEDSAVYYCA,TSEDSAVYYCARY, SAVYYCARYFFGS, AVYYCARYFFGSS, VYYCARYFFGSSP,CARYFFGSSPNWY, ARYFFGSSPNWYF, RYFFGSSPNWYFD, YFFGSSPNWYFDV,PNWYFDVWGAGTT, NWYFDVWGAGTTV, WYFDVWGAGTTVT, FDVWGAGTTVTVS,DVWGAGTTVTVSS

[0228] Peptide sequences in the light-chain variable region of antibody5G1.1 with potential human MHC class II binding activity are:IQMTQSPASLSAS, ASLSASVGETVTI, ASVGETVTITCGA, ETVTITCGASENI,VTITCGASENIYG, TITCGASENIYGA, ENIYGALNWYQRK, NIYGALNWYQRKQ,GALNWYQRKQGKS, LNWYQRKQGKSPQ, NWYQRKQGKSPQL, GKSPQLLIYGATN,PQLLIYGATNLAD, QLLIYGATNLADG, LLIYGATNLADGM, LIYGATNLADGMS,TNLADGMSSRFSG, DGMSSRFSGSGSG, SRFSGSGSGRQYY, SGSGRQYYLKISS,RQYYLKISSLHPD, QYYLKISSLHPDD, YYLKISSLHPDDV, LKISSLHPDDVAT,SSLHPDDVATYYC, SLHPDDVATYYCQ, DDVATYYCQNVLN, ATYYCQNVLNTPL,TYYCQNVLNTPLT, YYCQNVLNTPLTF, YCQNVLNTPLTFG, CQNVLNTPLTFGA,QNVLNTPLTFGAG, NVLNTPLTFGAGT, TPLTFGAGTKLEL

EXAMPLE 19 Anti-CD20 Antibodies

[0229] The present invention provides for modified forms of a monoclonalantibody with binding specificity to the human CD20 antigen. CD20 is aB-cell specific surface molecule expressed on pre-B and mature B-cellsincluding greater than 90% of B-cell non-Hodgkin's lymphomas (NHL).Monoclonal antibodies and radioimmunoconjugates targeting of CD20 haveemerged as new treatments for NHL. Significant examples include themonoclonal antibodies 2B8 [Reff, M. E. et al (1994) Blood 83: 435-445]and B1 [U.S. Pat. No. 6,090,365]. The variable region domains of 2B8have been cloned and combined with human constant region domains toproduce a chimeric antibody designated C2B8 which is marketed asRituxan™ in the USA [U.S. Pat. No. 5,776,456] or MabThera^(R)(rituximab) in Europe. C2B8 is recognized as a valuable therapeuticagent for the treatment of NHL and other B-cell diseases [Maloney, D. G.et al (1997) J. Clin. Oncol. 15: 3266-3274; Maloney, D. G. et al (1997)Blood 90: 2188-2195]. The B1 antibody has similarly achievedregistration for use as a NHL therapeutic although in this case themolecule (Bexxar™) is a ¹³¹I radioimmunoconjugate although the native(non-conjugated) antibody has utility in ex vivo purging regimens forautologous bone marrow transplantation therapies for lymphoma andrefractory leukemia [Freedman, A. S. et al (1990), J. Clin. Oncol. 8:784]. Despite the success of antibodies such as C2B8 (rituximab) andBexxar™ there is a continued need for anti-CD20 analogues with enhancedproperties. Peptide sequences in the heavy-chain variable region ofantibody 2B8 with potential human MHC class II binding activity are:VQLQQPGAELVKA, LQQPGAELVKAGA, AELVKAGASVKMS, ELVKAGASVKMSC,ASVKMSCKASGYT, VKMSCKASGYTFT, KMSCKASGYTFTS, ASGYTFTSYNMHW,SGYTFTSYNMHWV, YTFTSYNMHWVKQ, TSYNMHWVKQTPG, YNMHWVKQTPGRG,MHWVKQTPGRGLE, HWVKQTPGRGLEW, TPGRGLEWIGAIY, RGLEWIGAIYPGN,GLEWIGAIYPGNG, EWIGAIYPGNGDT, GAIYPGNGDTSYN, AIYPGNGDTSYNQ,YPGNGDTSYNQKF, TSYNQKFKGKATL, YNQKFKGKATLTA, QKFKGKATLTADK,ATLTADKSSSTAY, TAYMQLSSLTSED, AYMQLSSLTSEDS, MQLSSLTSEDSAV,SSLTSEDSAVYYC, SLTSEDSAVYYCA, TSEDSAVYYCARS, SAVYYCARSTYYG,AVYYCARSTYYGG, VYYCARSTYYGGD, STYYGGDTYFNVW, TYYGGDTYFNVWG,DTYFNVWGAGTTV, TYFNVWGAGTTVT, FNVWGAGTTVTVS, NVWGAGTTVTVSA

[0230] Peptide sequences in the light-chain variable region of antibody2B8 with potential human MHC class II binding activity are:QIVLSQSPAILSA, IVLSQSPAILSAS, QSPAILSASPGEK, PAILSASPGEKVT,AILSASPGEKVTM, EKVTMTCRASSSV, VTMTCRASSSVSY, TMTCRASSSVSYI,SSVSYIHWFQQKP, VSYIHWFQQKPGS, SYIHWFQQKPGSS, IHWFQQKPGSSPK,KPWIYATSNLASG, PWIYATSNLASGV, WIYATSNLASGVP, ATSNLASGVPVRF,SNLASGVPVRFSG, SGVPVRFSGSGSG, VPVRFSGSGSGTS, VRFSGSGSGTSYS,GTSYSLTISRVEA, TSYSLTISRVEAE, SYSLTISRVEAED, YSLTISRVEAEDA,LTISRVEAEDAAT, SRVEAEDAATYYC, RVEAEDAATYYCQ, ATYYCQQWTSNPP,TYYCQQWTSNPPT, QQWTSNPPTFGGG, NPPTFGGGTKLEI

EXAMPLE 20

[0231] The present invention provides for modified forms of a monoclonalantibody with binding specificity to the human IL-2 receptor. Themonoclonal antibody is designated anti-Tac and the modified form has oneor more T cell epitopes removed. The anti-Tac antibody binds with highspecificity to the alpha subunit (p55-alpha, CD25 or Tac) of the humanhigh affinity IL-2 receptor expressed on the surface of T and Blymphocytes. Antibody binding blocks the ability of IL-2 to bind thereceptor and achieve T-cell activation. The ability of the anti-Tacantibody to act as an IL-2 antagonist has significant clinical potentialin the treatment of organ transplant rejection. Clinical studies usingthe mouse antibody have shown some initial benefit to patients who haveundergone kidney transplant although the long term benefit overconventional immune suppression was not found due the development of aHAMA response in a high proportion of patients [Kirkham, R. L. et al(1991) Transplantation 51: 107-113]. A “humanized” anti-Tac antibody hasbeen developed in which significant components of the protein have beenengineered to contain protein sequence identified from a human antibodygene [Queen, C. et al (1989) Proc. Natl. Acad. Sci. (USA) 86:10029-10033; U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat.No. 6,013,256]. The “humanised” anti-Tac (Zenapax™ or daclizumab) hasundergone clinical trials as an immune suppressive agent for themanagement of acute graft versus host disease and suppression of kidneytransplant rejection [Anasetti, C. et al (1994), Blood 84: 1320-1327;Anasetti, C. et al (1995) Blood 86: Supplement 1:62a; Eckhoff, D. E. etal (2000) Transplantation 69: 1867-1872; Ekberg, H. et al (1999)Transplant Proc. 31: 267-268].

[0232] Peptide sequences in the heavy-chain variable region of mouseanti-Tac antibody with potential human MHC class II binding activityare: VQLQQSGAELAKP, AELAKPGASVKMS, ASVKMSCKASGYT, VKMSCKASGYTFT,KMSCKASGYTFTS, ASGYTFTSYRMHW, SGYTFTSYRMHWV, YTFTSYRMHWVKQ,TSYRMHWVKQRPG, YRMHWVKQRPGQG, MHWVKQRPGQGLE, HWVKQRPGQGLEW,RPGQGLEWIGYIN, QGLEWIGYINPST, LEWIGYINPSTGY, EWIGYINPSTGYT,IGYINPSTGYTEY, GYINPSTGYTEYN, TGYTEYNQKFKDK, TEYNQKFKDKATL,QKFKDKATLTADK, ATLTADKSSSTAY, TAYMQLSSLTFED, AYMQLSSLTFEDS,YMQLSSLTFEDSA, MQLSSLTFEDSAV, SSLTFEDSAVYYC, SLTFEDSAVYYCA,LTFEDSAVYYCAR, SAVYYCARGGGVF, AVYYCARGGGVFD, VYYCARGGGVFDY,GGVFDYWGQGTTL, GVFDYWGQGTTLT, FDYWGQGTTLTVS, DYWGQGTTLTVSS

[0233] Peptide sequences in the light-chain variable region of mouseanti-Tac antibody with potential human MHC class II binding activityare: QIVLTQSPAIMSA, IVLTQSPAIMSAS, QSPAIMSASPGEK, PAIMSASPGEKVT,AIMSASPGEKVTI, EKVTITCSASSSI, VTITCSASSSISY, TITCSASSSISYM,SSISYNHWFQQKP, ISYMHWFQQKPGT, SYMHWFQQKPGTS, MHWFQQKPGTSPK,HWFQQKPGTSPKL, SPKLWIYTTSNLA, PKLWIYTTSNLAS, KLWIYTTSNLASG,LWIYTTSNLASGV, WIYTTSNLASGVP, TTSNLASGVPARF, SNLASGVPARFSG,SGVPARFSGSGSG, ARFSGSGSGTSYS, GTSYSLTISRMEA, TSYSLTISRMEAE,SYSLTISRMEAED, YSLTISRMEAEDA, LTISRMEAEDAAT, SRMEAEDAATYYC,ATYYCHQRSTYPL, TYYCHQRSTYPLT, STYPLTFGSGTKL, TYPLTFGSGTKLE,YPLTFGSGTKLEL

[0234] Peptide sequences in the heavy-chain variable region of humanizedanti-Tac antibody with potential human MHC class II binding activityare: VQLVQSGAEVKKP, QLVQSGAEVKKPG, AEVKKPGSSVKVS, SSVKVSCKASGYT,VKVSCKASGYTFT, KVSCKASGYTFTS, ASGYTFTSYRMHW, SGYTFTSYRMHWV,YTFTSYRMHWVRQ, TSYRMHWVRQAPG, YRMHWVRQAPGQG, MHWVRQAPGQGLE,HWVRQAPGQGLEW, RQAPGQGLEWIGY, APGQGLEWIGYIN, QGLEWIGYINPST,LEWIGYINPSTGY, EWIGYINPSTGYT, WIGYINPSTGYTE, IGYINPSTGYTEY,GYINPSTGYTEYN, TGYTEYNQKFKDK, TEYNQKFKDKATI, QKFKDKATITADE,ATITADESTNTAY, TITADESTNTAYM, TNTAYMELSSLRS, TAYMELSSLRSED,AYMELSSLRSEDT, MELSSLRSEDTAV, SSLRSEDTAVYYC, SLRSEDTAVYYCA,RSEDTAVYYCARG, TAVYYCARGGGVF, AVYYCARGGGVFD, VYYCARGGGVFDY,GGVFDYWGQGTLV, GVFDYWGQGTLVT, FDYWGQGTLVTVS, DYWGQGTLVTVSS

[0235] Peptide sequences in the light-chain variable region of humanizedanti-Tac antibody with potential human MHC class II binding activityare: IQMTQSPSTLSAS, STLSASVGDRVTI, ASVGDRVTITCSA, DRVTITCSASSSI,VTITCSASSSISY, TITCSASSSISYM, SSISYMHWYQQKP, ISYMHWYQQKPGK,SYMHWYQQKPGKA, MHWYQQKPGKAPK, HWYQQKPGKAPKL, QKPGKAPKLLIYT,PKLLIYTTSNLAS, KLLIYTTSNLASG, LLIYTTSNLASGV, LIYTTSNLASGVP,TTSNLASGVPARF, SNLASGVPARFSG, SGVPARFSGSGSG, ARFSGSGSGTEFT,SGSGTEFTLTISS, GTEFTLTISSLQP, TEFTLTISSLQPD, FTLTISSLQPDDF,LTISSLQPDDFAT, TISSLQPDDFATY, SSLQPDDFATYYC, SLQPDDFATYYCH,DDFATYYCHQRST, ATYYCHQRSTYPL, TYYCHQRSTYPLT, STYPLTFGQGTKV,TYPLTFGQGTKVE, YPLTFGQGTKVEV

EXAMPLE 21 14.18 Antibody

[0236] Unless stated otherwise all amino acids in the variable heavy andlight chains are numbered as in Kabat et al., 1991 (Sequences ofProteins of Immunological Interest, US Department of Health and HumanServices). Potential T-cell epitopes are numbered with the linear numberof the first amino acid of an epitope, counting from the first aminoacid of the heavy and light chains.

[0237] 1 Comparison with Mouse Subgroup Frameworks

[0238] The amino acid sequences of murine 14.18 VH and VK were comparedto consensus sequences for the Kabat murine heavy and light chainsubgroups (Kabat et al., 1991). 14.18 VH can be assigned to Mouse HeavyChains Subgroup II(A). The sequence of 14.18VH is shown in SEQ No. 1.The comparison with the consensus sequence of this subgroup shows thatthe histidine at position 81 (normally glutamine), the lysine atposition 82a (normally serine or asparagine), the valine at position 93(normally alanine) and the serine at position 94 (normally arginine) area typical for this subgroup. The residues at positions 19, 40 and 66 arealso found infrequently in this subgroup, but are considered to haveminor effects on antibody binding and structure. 14.18 VK can beassigned to Mouse Kappa Chains Subgroup II. The comparison to theconsensus sequence for this subgroup shows that the histidine atposition 49 is a typical for this subgroup. This residue is mostcommonly tyrosine.

[0239] 2 Comparison with Human Frameworks

[0240] The amino acid sequences of murine 14.18 V_(H) and V_(K) werecompared to the sequences of the directories of human germline V_(H)(Tomlinson et al., J. Mol. Biol. 1992: 227, 776-798) and VK (Cox et. al.(Eur. J. Immunol. 1994; 1-4-.827-36)) sequences and also to humangermline J region sequences (Routledge et al., In “Protein Engineeringof Antibody Molecules for Prophylactic and Therapeutic Applications inMan”. Clark M ed. Academic Titles, Nottingham pp. 13-44, 1993). Thereference human framework selected for 14.18 V_(H) was DP25 with humanJ_(H)6. This germline sequence has been found in a rearranged matureantibody gene with no amino acid changes. For framework 3 the sequenceof the mature human antibody 29 was used. This sequence is identical tothe murine sequence immediately adjacent to CDR3. The reference humanframework selected for 14.18 VK was DPK22. This germline sequence hasbeen found in a rearranged mature antibody gene with no amino acidchanges. For framework 2 the sequence of the mature human antibody 163.5was used. This sequence is identical to the murine sequence immediatelyadjacent to CDR2. The J region sequence was human JK2 (Routledge et al.,1993).

[0241] 3 Design of Veneered Sequences

[0242] Following identification of the reference human frameworksequences, certain non-identical amino acid residues within the 14.18V_(H) and V_(K) frameworks were changed to the corresponding amino acidin the human reference sequence. Residues which are considered to becritical for antibody structure and binding were excluded from thisprocess and not altered. The murine residues that were retained at thisstage are largely non-surface, buried residues, apart from residues atthe N-terminus for instance, which are close to the CDRs in the finalantibody. This process produces a sequence that is broadly similar to a“veneered” antibody as the surface residues are mainly human and theburied residues are as in the original murine sequence.

[0243] 4 Peptide Threading Analysis

[0244] The murine and veneered 14.18 V_(H) and V_(K) sequences wereanalyzed using the method according to the invention. The amino acidsequences are divided into all possible 13-mers. The 13-mer peptides aresequentially presented to the models of the binding groove of the HLA-DRallotypes and a binding score assigned to each peptide for each allele.A conformational score is calculated for each pocket-bound side chain ofthe peptide. This score is based on steric overlap, potential hydrogenbonds between peptide and residues in the binding groove, electrostaticinteractions and favorable contacts between peptide and pocket residues.The conformation of each side chain is then altered and the scorerecalculated. Having determined the highest conformational score, thebinding score is then calculated based on the groove-bound hydrophobicresidues, the non-groove hydrophilic residues and the number of residuesthat fit into the binding groove. Known binders to NMC class II achievea significant binding score with almost no false negatives. Thuspeptides achieving a significant binding score from the current analysisare considered to be potential T-cell epitopes. The results of thepeptide threading analysis for the murine and veneered sequences areshown in Table 1. TABLE 1 Potential T-cell epitopes in murine andveneered 14.18 sequences Number of potential Sequence T-cell Location ofpotential epitopes Murine 14, 18 VH 11 3(17), 9(15), 30(5), 35(17),39(15), 43(9), 58(12), 62(11), 81(11), 84(16), 101(7) Veneered 14. 18 543(9), 58(12), 62(11), 81(11), 84(16) VH Murine 14.18 VK 7 7(7), 13(11),27(15), 49(11), 86(17), 97(11), 100(4) Veneered 14. 18 5 27(I5), 49(11),86(17), 97(11), 100(17) VK

[0245] 5 Removal of Potential T Cell Epitopes

[0246] Potential T-cell epitopes are removed by making amino acidsubstitutions in the particular peptide that constitutes the epitope.Substitutions were made by inserting amino acids of similarphysicochemical properties if possible. However in order to remove somepotential epitopes, amino acids of different size, charge orhydrophobicity may need to be substituted. IT changes have to madewithin CDRs which might have an effect on binding, it is necessary tomake a variant with and without the particular amino acid substitution.The linear number for amino acid residues for substitution is given withthe Kabat number in brackets. Potential T Cell epitopes are referred toby the linear number of the first residue of the 13-mer. The amino acidchanges required to remove T-cell epitopes from the veneered 14.18 heavychain variable region were as follows:

[0247] 1 Substitution of isoleucine for proline at residue 41 (Kabatnumber 41), combined with substituting leucine for alanine at residue 50in CDR2 removes the potential epitope at position 43.

[0248] 2 An alternative to (1), substitution of threonine for leucine atresidue 45 (Kabat number 45) with proline at position 41 (Kabat number41) also removes the potential epitope at position 43.

[0249] 3 Substitution of serine for glycine at residue 66 (Kabat number65) in CDR2 and valine for alanine at residue 68 (Kabat number 67)removes the potential epitope at position 58. Serine is found at thisposition in human and mouse antibody sequences.

[0250] 4 Substitution of isoleucine for leucine at residue 70 (Kabat:69) reduces the number of MHC allotypes that bind to the potentialepitope at position 62 from 11 to 4.

[0251] 5 Substitution of alanine for valine position 72 (Kabat number71) removes the potential epitope position 62. The size of the aminoacid at this position is critical and alanine is similar in size andhydrophobicity to valine.

[0252] 6. Substitution of threonine for serine at residue 91 (Kabatnumber 87) removes the potential epitopes at positions 81 and 84.

[0253] The amino acid substitutions required to remove the potentialT-cell epitopes from the veneered 14.18 light chain variable region wereas follows:

[0254] 1. Substitution of serine for arginine at residue 32 (Kabatnumber 27e) removes the potential epitope at position 27. This residueis within CDR2, however serine is often found at tWs position in mouseand human antibodies. There is no change outward the CDR which removesthis potential T-cell epitope.

[0255] 2. Substitution of tyrosine for histidine at position 54 (Kabatnumber 49) eliminates the potential epitope at position 43. Tyrosine isthe most frequent amino acid found at position 49 in mouse and humanantibodies.

[0256] 3. An alternative change to (2) for elimination of the potentialepitope at position 43, is substitution of methionine for leucine atresidue 51 (Kabat number 46). Methionine is similar to leucine in sizeand hydrophobicity.

[0257] 4. Substitution of methionine for leucine at residue 88 (Kabatnumber 83) removes the potential epitope at position 86.

[0258] 5. Substitution of threonine for leucine at residue 102 (Kabatnumber 96) in CDRH3, when combined with glutamine to glycine at position105 (Kabat number 100) reduces the number of MHC allotypes that bind tothe potential epitope at position 97 from 11 to 5.

[0259] 6. An alternative change to (5) which eliminates the potentialepitope at position 97 is substitution of proline for leucine at residue102 (Kabat number 96).

[0260] 7. Substitution of valine for leucine at residue 110 (Kabatnumber 104) removes the potential epitope at position 100.

[0261] 6 Design of De-Immunized Sequences

[0262] De-immunized heavy and light chain sequences were designed withreference to the changes required to remove potential T-cell epitopesand consideration of framework residues that might be critical forantibody structure and binding. In addition to the De-immunizedsequences based on the veneered sequence, an additional sequence wasdesigned for each VH and VK based on the murine sequence, termed theMouse Peptide Threaded (MoPT) version. For this version, changes weremade directly to the murine sequence in order to eliminate T-cellepitopes, but only changes outside the CDRs that are not considered tobe detrimental to binding are made. No attempt to remove surface (Bcell) epitopes has been made in this version of the de-immunizedsequence.

[0263] The primary de-immunized VH includes substitutions 1, 3, 4, 5,and 6 in Section 5 above and includes no potential T-cell epitopes. Afurther 4 de-immunized VHS were designed in order to test the effect ofthe various substitutions required on antibody binding. Version 2 is analternative to Version 1 in which an alternative substitution (2 inSection 2.5 above) has been used to remove the same potential T-cellepitope. The cumulative alterations made to the primary de-immunizedsequence (14.18DIVH1) and the potential T-cell epitopes remaining aredetailed in Table 2. The mouse threaded version is included forcomparison. Table 2: Amino acid changes and potential epitopes inde-immunized 14.18 VH Potential epitopes Cumulative residue (no. ofpotential MHC Variant changes binders from 18 tested) 14.18DIVH1 nonenone 14.18DIVH2 41I → P, 45L → T, 50L → A none 14.18DIVH3 65S → G 58(8)14.18DIVH4 71A → V 58(8), 62(4) 14.18DIVH5 45T → L, 41P → 1 43(9) 58(8)62(4) 14.18MoPTVH NA 43(9) 58(12) 62(11)

[0264] The primary de-immunized VK includes substitutions 1, 2, 4, 6 and7 in Section 5 above. The primary de-immunized VK includes no potentialT-cell epitopes. A further 5 De-immunized VKS were designed in order totest the effect of the various substitutions required on antibodybinding. Version 2 is an alternative to Version 1 in which a differentsubstitution has been used to remove the potential T-cell epitope atposition 43. Versions 3 includes the alternative substitution (6 inSection 2.5 above), which reduces the number of MHC allotypes that bindto the potential epitope at position 97 from 11 to 5. The cumulativealterations made to the primary De-immunized sequence (14.18DIVK1) andthe potential T-cell epitopes remaining are detailed in Table 3. TABLE 3Amino acid changes and potential epitopes in de-immunized 14.18 VKPotential epitopes' (no. Cumulative of potential MHC Variant residuechanges* binders from 18 tested) 14.18DIVKI None none 14.18DIVK2 46L →M, 49Y → H none 14.18DIVK3 96P → T, 100Q → G 97(5) 14.18DIVK4 96T → L97(11) 14.18DIVK5 27e S → R 27(15), 97(11) 14.18DIVK6 46M → L 27(15),49(11), 97(11) 14.18MoPTVK NA 27(15), 49(11), 97(11), 100(4)

[0265] Sequences of versions of modified epitopes: 14.18 VH veneered:EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRLEWIGAIDPYYGGTSYNQKFKGRATLSVDKSSSQAYMHLKSLTSEDSAVYYCVSGMEYWGQGTTVTVSS 14.18 VK veneered:DVVMTQSPGTLPVSLGERATISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDLAVYFCSQSTHVPPLTFGQGTKLEIK 14.18 de-immunized VH1EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAIGQRLEWIGLIDPYYGGTSYNQKFKSRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK1DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPPTFGQGTKVEIK 14.18 de-immunized VH2EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKSRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immimized VK2DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPPTFGQGTKVEIK 14.18 de-immunized VH3EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKGRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK3DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISPLEAEDMAVYFCSQSTHVPPTTFGGGTKVEIK 14.18 de-immunized VH4EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKGRVTITVDKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK4DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPLTFGGGTKVEIK 14.18 de-immunized VH5EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAIGQRLEWIGAIDPYYGGTSYNQKFKGRVTITVDKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK5DVVMTQSPGTLPVSLGERATISCRSSQSLVHRNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPLTFGGGTKVEIK 14.18 VH mouse, peptidethreaded (Mo PT)EVQLVQSGPEVEKPSASVKISCKASGSSFTGYNMNWVRQAIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 VK mouse, peptidethreaded (Mo PT)DVVMTQTPGSLPVSAGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDSGVYFCSQSTHVPPLTFGAGTKLELK 14.18 VH mouseEVQLLQSGPELEKPSASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS 14.18 VK mouseDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

EXAMPLE 22 KS Antibody

[0266] 1 Comparison with Mouse Subgroup Frameworks

[0267] The amino acid sequences of murine KS VH and VK were compared toconsensus sequences for the Kabat murine heavy and light chain subgroups(Kabat et al., 1991). Murine KS VH cannot be assigned to any oneSubgroup, but is closest to Subgroup II(A) and V(A). Unusual residuesare found at position 2 which is normally valine, 46 which is normallyglutamic acid, and 68 which is normally threonine. Residue 69 is morecommonly leucine or iso-leucine. At 82b, serine is most often found.Murine KS VK can be assigned to Subgroup VI (FIG. 2). Unusual residuesare found at 46 and 47 which are commonly both leucine. Residue 58 isunusual with either leucine or valine normally found at this position.

[0268] 2 Comparison with Human Frameworks

[0269] The amino acid sequences of murine KS VH and VK were compared tothe sequences of the directory of human germline VH (Tomlinson et al.,1992) and VK (COX et al. 1994) sequences and also to human germline Jregion sequences (Routledge et al., 1993). The reference human frameworkselected for KS VH was DP10 with human JH6. This germline sequence hasbeen found in a rearranged mature antibody gene with no amino acidchanges. The reference human framework selected for KS VK was B 1. Forframework-2 the sequence of the mature human antibody IMEV was used (inKabat et al 1991). This sequence is identical to the murine sequenceimmediately adjacent to CDR2. The J region sequence was human JK4. Thisgermline sequence has not been found as rearranged mature antibody lightchain.

[0270] 3 Design of Veneered Sequences

[0271] Following identification of the reference human frameworksequences, certain non-identical amino acid residues within the 425 VHand VK frameworks were changed to the corresponding amino acid in thehuman reference sequence. Residues which are considered to be criticalfor antibody structure and bindin2 were excluded from this process andnot altered. The murine residues that were retained at this stage arelargely non-surface, buried residues, apart from residues at theN-terminus for instance, which are close to the CDRs in the finalantibody. This process produces a sequence that is broadly similar to a“veneered” antibody as the surface residues are mainly human and theburied residues are as in the original murine sequence.

[0272] 4 Peptide Threading Analysis

[0273] The murine and veneered KS VH and VK sequences were analyzedusing the method according to the invention. The amino acid sequencesare divided into all possible 13imers. The 13-mer peptides aresequentially presented to the models of the binding groove of the HLA-DRallotypes and a binding score assigned to each peptide for each allele.A conformational score is calculated for each pocket-bound side chain ofthe peptide. This score is based on steric overlap, potential hydrogenbonds between peptide and residues in the binding groove, electrostaticinteractions and favorable contacts between peptide and pocket residues.The conformation of each side chain is then altered and the scorerecalculated. Having determined the highest conformational score, thebinding score is then calculated based on the (groove-bound hydrophobicresidues, the non-groove hydrophilic residues and the number of residuesthat fit into the binding groove. Known binders to MHC class II achievea significant binding score with almost no false negatives. Thuspeptides achieving, a significant binding score from the currentanalysis are considered to be potential T cell epitopes. The results ofthe peptide threading analysis for the murine and veneered sequences areshown in Table 1. TABLE 1 Potential T cell epitopes in murine andveneered KS sequences Number Location of potential of potential Tepitopes (no. of potential Sequence cell epitopes MHC binders) Murine KSVH 6 35(11), 62(17), 78(12), 81(12), 89(6), 98(15) Murine KS VH 5 30(7),62(15), 78(11), 89(6), 98(15) Murine KS VK 6 1(14), 2(5), 17(5), 27(5),51(13), 72(18) Veneered KS VK 3 1(17), 27(5), 51(13)

[0274] 5 Removal of Potential T Cell Epitopes

[0275] Potential T cell epitopes are removed by making amino acidsubstitutions in the particular peptide that constitutes the epitope.Substitutions were made by inserting amino acids of similarphysicochemical properties if possible. However in order to remove somepotential epitopes, amino acids of different size, charge orhydrophobicity may need to be substituted. If changes have to madewithin CDRs which might have an effect on binding, there is then a needto make a variant with and without the particular amino acidsubstitution. Numbering of amino acid residues for substitution is asper Kabat. Potential T Cell epitopes are referred to by the linearnumber of the first residue of the 13mer.

[0276] The amino acid changes required to remove T cell epitope from theveneered KS heavy chain variable region were as follows:

[0277] 1. Substitution of arginine for lysine at residue 38 (Kabatnumber 38) removes the potential epitope at residue no 30.

[0278] 2. Substitution of alanine for leucine at residue 72 (Kabatnumber 71) and isoleucine for phenylalanine at residue 70 (Kabat number69) removes the potential epitope at residue 62. An isoleucine at Kabatnumber 69 and alanine at Kabat number 71 is found in a human germline VHsequence, DP10.

[0279] 3. Substitution of leucine for alanine at residue 79 (Kabatnumber 78) removes the potential epitope at residue number 78.

[0280] 4. Substitution of threonine for methionine at residue 91 (Kabatnumber 87), removes the potential epitope at residue number 89.

[0281] 5. Substitution of methionine for at isoleucine residue 100(Kabat number 96) in CDRH3 removes the potential epitope at residue 98.There is no change out with CDRH3 which removes this potential epitope.

[0282] The amino acid substitutions required to remove the potential Tcell epitopes from the veneered KS light chain variable region were asfollows:

[0283] 1. Substitution of isoleucine for methionine at residue 32 (Kabatnumber 33) removes the potential epitope at residue number 27. Thisresidue is within CDR2. Isoleucine is commonly found at this position inhuman antibodies.

[0284] 2. The potential epitope at position 1 is removed by substitutingvaline for leucine at residue (Kabat number 3).

[0285] 3. Substitution of serine for alanine at residue 59 (Kabat number60) removes the potential epitope at residue number 51.

[0286] 6 Design of De-Immunized Sequences

[0287] De-immunized heavy and light chain sequences were designed withreference to the changes required to remove potential T cell epitopesand consideration of framework residues that might be critical forantibody structure and binding. In addition to the de-immunizedsequences based on the veneered sequence, an additional sequence wasdesigned for each VH, and VK based on the murine sequence, termed theMouse Peptide Threaded (MOPT) version. For this version, changes, weremade directly to the murine sequence in order to eliminate T cellepitopes, but only changes outside the CDRs that are not considered tobe detrimental to binding are made. No attempt to remove surface (Bcell) epitopes has been made in this version of the de-immunizedsequence. The primary de-immunized VH includes substitutions 1 to 5 inSection 5 above and one extra change at residue 43 (Kabat number 43).Lysine found in the murine sequence was substituted for the glutaminefrom the human framework. Lysine is positively charged and thereforesignificantly different to glutamine; this region may be involved inVH/VL contacts. The primary de-immunized VH includes no potential T cellepitopes. A further 4 de-immunized VHs were designed in order to testthe effect of the various substitutions required on antibody binding.The cumulative alterations made to the primary de-immunized sequence(KSDIVHv1) and the potential T cell epitopes remaining are detailed inTable 2. TABLE 2 Amino acid changes and potential epitopes inde-immunized KS VH Potential epitopes (no. of potential Cumulativeresidue MHC binders Variant changes from 18 tested) KSDIVHv1 None noneKSDIVHv2 96M → I 98(15) KSDIVHv3 71A → L, 78L → A 62(16), 78(11), 98(15)KSDIVHv4 38 R → K 30(7), 62(16), 78(11), 98(15) KSDIVHv5 68T → A, 69I →F 30(7), 62(17), 78(11), 98(15) KSMoPTVH NA 98(15), 78(12)

[0288] The primary de-immunized VK includes substitutions 1 to 3 inSection 5 above. A further 3 de-immunized VKs were designed in order totest the effect of the various substitutions required on antibodybinding. The cumulative alterations made to the primary de-immunizedsequence (KSDIVKv1) and the potential T cell epitopes remaining aredetailed in Table 3. TABLE 3 Amino acid changes and potential epitopesin de-immunized KS VK Potential epitopes Cumulative residue (no. ofpotential MHC binders Variant changes from 18 tested) KSDIVKv1 None noneKSDIVKv2 33I → M 27(5) KSDIVKv3 3V → L 1(17), 27(5) KSDIVKv4 60 S → A1(17), 27(5), 5(13) KSMoPTVK NA none

[0289] Sequences of versions of modified epitopes: KS VH veneered:QIQLVQSGPELKKPGSSVKiSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFKGRFTfT1ETSTSTAYLQLNNLRsEDmATYfCVRFISKGDYWGQGTTVTVSS KS VK veneered:QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPARFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immnunized VH1QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITAETSTSTLYLQLNNLRSEDTATYFCVRFMSKGDYWGQGTTVTVSS KS de-immunized VK1QIVLTQSPASLAVSPGQRATITCSASSSVSYILWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH2QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITAETSTSTLYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK2QIVLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH3QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK3QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH4QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK4QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPARFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH5QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFTLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK5QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGSSPKPWIYDTSNLASGFPARFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS VH mouse, peptide threaded (MoPT)QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAFLQLNNLRSEDTATYFCVRFISKGDYWGQGTSVTVSS KS VK mouse, peptidethreaded (Mo PT)QIVLTQSPATLSASPGERVTITCSASSSVSYMLWYLQKPGSSPKPWIFDTSNLASGFPSRFSGSGSGTTYSLIISSLEAEDAATYYCHQRSGYPYTFGGGTKLEIK KS VH mouseQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQTPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAFLQINNLRNEDMATYFCVRFISKGDYWGQGTSVTVSS KS VK mouseQILLTQSPAIMSASPGEKVTMTCSASSSVSYMLWYQQKPGSSPKPWIFDTSNLASGFPARFSGSGSGTSYSLIISSMEAEDAATYYCHQRSGYPYTFGGGTKLEIK

[0290]

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040180386). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A method suitable for identifying one or more potential T-cellepitope peptides within the amino acid sequence of a biological moleculeby steps including determination of the binding of said peptides to MHCmolecules using in vitro or in silico techniques or biological assays,said method comprises the following steps: (a) selecting a region of thepeptide having a known amino acid residue sequence; (b) sequentiallysampling overlapping amino acid residue segments of predetermineduniform size and constituted by at least three amino acid residues fromthe selected region; (c) calculating MHC Class II molecule binding scorefor each said sampled segment by summing assigned values for eachhydrophobic amino acid residue side chain present in said sampled aminoacid residue segment; and (d) identifying at least one of said segmentssuitable for modification, based on the calculated MHC Class II moleculebinding score for that segment, to change overall MHC Class II bindingscore for the peptide without substantially the reducing therapeuticutility of the peptide.
 2. The method according to claim 1, wherein step(c) is carried out by using a Böhm scoring function modified to include12-6 van der Waal's ligand-protein energy repulsive term and ligandconformational energy term by (1) providing a first data base of MHCClass II molecule models; (2) providing a second data base of allowedpeptide backbones for said MHC Class II molecule models; (3) selecting amodel from said first data base; (4) selecting an allowed peptidebackbone from said second data base; (5) identifying amino acid residueside chains present in each sampled segment; (6) determining the bindingaffinity value for all side chains present in each sampled segment; andoptionally (7) repeating steps (1) through (5) for each said model andeach said backbone.
 3. The method of claim 1 or 2, wherein the assignedvalue for each aromatic side chain is about one-half of the assignedvalue for each hydrophobic aliphatic side chain.
 4. The method of any ofthe claims 1-3, wherein the sampled amino acid residue segment isconstituted by 13 amino acid residues.
 5. The method of any of theclaims 1-4, wherein consecutive sampled amino acid residue segmentsoverlap by one to five amino acid residues.
 6. The method of any of theclaims 1-4, wherein consecutive sampled amino acid residue segmentsoverlap one another substantially.
 7. The method of any of the claims1-4, wherein all but one of amino acid residues in consecutive sampledamino acid residue segments overlap.
 8. A method for preparing animmunogenicly modified biological molecule derived from a parentmolecule, wherein the modified molecule has an amino acid sequencedifferent from that of said parent molecule and exhibits a reducedimmunogenicity relative to the parent molecule when exposed to theimmune system of a given species; said method comprises: (i) determiningthe amino acid sequence of the parent biological molecule or partthereof; (ii) identifying one or more potential T-cell epitopes withinthe amino acid sequence of the protein by any method includingdetermination of the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays, (iii) designing newsequence variants by alteration of at least one amino acid residuewithin the originally identified T-cell epitope sequences, said variantsare modified in such a way to substantially reduce or eliminate theactivity or number of the T-cell epitope sequences and/or the number ofMHC allotypes able to bind peptides derived from said biologicalmolecule as determined by the binding of the peptides to MHC moleculesusing in vitro or in silico techniques or biological assays or bybinding of peptide-MHC complexes to T-cells, (iv) constructing suchsequence variants by recombinant DNA techniques and testing saidvariants in order to identify one or more variants with desirableproperties, and (v) optionally repeating steps (ii)-(iv), characterizedin that the identification of T-cell epitope sequences according to step(ii) is achieved by a method as specified in any of the claims 1-7. 9.The method of claim 8, wherein 1-9 amino acid residues in any of theoriginally present T-cell epitope sequences are altered.
 10. The methodaccording to claim 9, wherein one amino acid residues in any of theoriginally present T-cell epitope sequences is altered.
 11. The methodof claim 8, wherein the amino acid alteration is made with reference toan homologous protein sequence.
 12. The method of claim 8, wherein theamino acid alteration is made with reference to in silico modelingtechniques.
 13. The method of any of the claims 8-12, wherein thealteration of the amino acid residues is substitution, deletion oraddition of originally present amino acid(s) residue(s) by other aminoacid residue(s) at specific position(s).
 14. The method of any of theclaims 8-13, wherein additionally further alteration is conducted torestore biological activity of said biological molecule.
 15. The methodof claim 14, wherein the additional further alteration is substitution,addition or deletion of specific amino acid(s).
 16. The method accordingto any of the claims 8-15, for preparing a polypeptide, a protein, afusion protein, an antibody or a fragment thereof with reducedimmunogenicity.
 17. The method of claim 16, wherein said polypeptide,protein, fusion protein, or antibody is selected from the groups: (a)monoclonal antibodies: anti-40 kD glycoprotein antigen antibody KS ¼,anti-GD2 antibody 14.18 anti-Her2 antibody 4D5 (murine) and humanizedversion (Herceptin®), anti-IL-2R (anti-Tac) antibody (Zenapax®),anti-CD52 antibody (CAMPATH®); anti-CD20 antibodies (C2B8, Rituxan®;Bexxar®) antibody directed to the human C5 complement protein (b)proteins: sTNF-R1, sTNF-R2, sTNFR-Fc (Enbrel®), protein C, aerp30, ricinA, CNTFR ligands, subtilisin, GM-CSF, human follicle stimulating hormone(h-fsh) δ-glucocerebrosidase, GLP-1, apolipoprotein A1.
 18. Animmunogenicly modified biological molecule derived from a parentmolecule, wherein the modified molecule has an amino acid sequencedifferent from that of said parent molecule and exhibits a reducedimmunogenicity relative to the parent molecule when exposed to theimmune system of a given species, obtained by a method of any of theclaims 1-17.
 19. Use of a potential T-cell epitope peptide within theamino acid sequence of a parent immunogenicly non-modified biologicalmolecule identified according to any of the methods of claims 1-7 forpreparing a biological molecule with reduced immunogenicity and having aretained desired biological activity.
 20. Use a potential T-cell epitopepeptide according to claim 19, wherein said T-cell epitope is a 13merpeptide.
 21. Use of a peptide sequence consisting of at least 9consecutive amino acid residues of a 13mer T-cell epitope as specifiedin claim 19 for preparing a biological molecule with reducedimmunogenicity as compared with the parent non-modified molecule andhaving biological activity.